Osteopontin antibodies

ABSTRACT

The present disclosure provides isolated antibodies, particularly human antibodies, or antigen binding portions thereof, that bind to osteopontin with high affinity. Nucleic acid molecules encoding the antibodies of the disclosure, expression vectors, host cells and methods for expressing the antibodies of the disclosure are also provided. 
     Immunoconjugates, bispecific molecules and pharmaceutical compositions comprising the antibodies or antigen binding portions thereof are also provided. The disclosure also provides methods for treating various cancers using the anti-osteopontin antibodies or antigen binding portions thereof described herein.

RELATED APPLICATIONS

This application claims the benefit of U.S. Patent Application No.61/235,542, filed Aug. 20, 2009, which is hereby incorporated byreference in its entirety.

REFERENCE TO SEQUENCE LISTING

This application is being filed electronically via EFS-Web and includesan electronically submitted sequence listing in .txt format. The .txtfile contains a sequence listing entitled “PC33873A_SequenceListing.txt”created on Aug. 19, 2010 and having a size of 81 KB. The sequencelisting contained in this .txt file is part of the specification and isincorporated herein by reference in its entirety.

FIELD

The present disclosure relates to antibodies and antigen-bindingportions thereof that bind to osteopontin. The disclosure also relatesto nucleic acid molecules encoding such antibodies and antigen-bindingportions, methods of making osteopontin antibodies and antigen-bindingportions, compositions comprising these antibodies and antigen-bindingportions, and methods of using the antibodies, antigen-binding portions,and compositions.

BACKGROUND

The human osteopontin (also known as SPP1) gene encodes a 314 amino acidresidue precursor protein with a 16 amino acid residue predicted signalpeptide that is cleaved to yield a 298 amino acid residue mature proteinwith an integrin binding sequence and N- and O-glycosylation sites.Osteopontin (OPN) is a secreted glycosylated phosphoprotein with amolecular weight between 44 and 75 kDa depending on posttranslationalmodifications of phosphorylation and/ or sulphation (Sodek et al., Crit.Rev. Oral Biol. Med. 11(3):279-303 (2000)). OPN contains the classic RGDmotif that is known to play a key role in cell attachment. The role ofOPN in bone is well-known in the art. Osteoclasts, which are thepredominant bone resorbing cell type, express the integrin αvβ3, amembrane-associated receptor for OPN (Dodds et al., J. Bone Miner. Res.10(11):1666-1680 (1995)). OPN is capable of binding to several celltypes including osteoblasts, osteoclasts, non transformed calvaria celllines and many transformed fibroblast cell lines (Somerman et al.,Matrix 9(1):49-54 (1989)). It has also been reported that OPN associateswith fibronectin (Singh et al., J. Biol. Chem. 265(30):18696-18701(1990); Nemir et al., J. Biol. Chem. 264(30):18202-18208 (1989)), type Icollagen (Chen et al., J. Biol. Chem. 267(34):24871-24878 (1992)), andosteocalcin (Ritter et al., J. Bone Miner. Res. 7(8):877-885 (1992)).

Aside from cell attachment, OPN can also affect cell physiology byinteraction with its receptor in a calcium dependent manner. OPN iscapable of binding multiple Ca²⁺ ions with relatively low affinity, andthe conformation of OPN is highly sensitive to changes in theconcentration of free Ca²⁺. The high density of negative charges aroundthe RGD cell binding sequence suggests that folding of the protein inthat region is dependent on free calcium levels, suggesting that calciummay affect the interaction of OPN with integrins.

OPN expression is also known to play a major role in malignantcarcinomas. Initially it was suggested that macrophages infiltrating thetumor, rather than the tumor cells themselves, express OPN (Furger etal., Curr. Mol. Med. 1(5):621-632 (2001)). More recently, however,certain tumor cells have been reported to directly express OPN (Rittlinget al., Br. J. Cancer 90(10):1877-1881 (2004)). Elevated levels of OPNwere detected in malignant breast tumors (Bellahcene et al., Am. J.Pathol. 146(1):95-100 (1995)), malignant glioblastomas (Takano et al.,Br. J. Cancer 82(12):1967-1973 (2000)), invasive primary cutaneousmelanoma (Zhou et al., J. Invest. Dermatol. 124(5):1044-1052 (2005)) andovarian cancer (Brakora et al., Gynecol. Oncol. 93(2):361-365 (2004))and strongly correlate with poor patient survival (Bramwell et al.,Clin. Cancer Res. 12(11):3337-3343 (2006).

SUMMARY

It is an object of the disclosure to provide human, or humanizedantibodies that specifically bind osteopontin. It is another object ofthe disclosure to provide antibodies that are safe for humanadministration. It is also an object of the present disclosure toprovide methods for treating disease and/or conditions associated withosteopontin up-regulation by using one or more antibodies of thedisclosure. These and other objects of the disclosure are more fullydescribed herein.

In one aspect, the disclosure provides an isolated human antibody orantigen-binding portion thereof that specifically binds osteopontin witha K_(D) of 600 nM or less, 100 nM or less, 50 nM or less, 10 nM or less,5 nM or less, or 1 nM or less. In one aspect, said osteopontin is humanosteopontin. In another aspect, said osteopontin is murine osteopontin.

In a further aspect, the disclosure provides an isolated antibody, orantigen-binding portion thereof, that specifically binds osteopontin,wherein said antibody or antigen-binding portion comprises: (a) anH-CDR1 as set forth in SEQ ID NO:1, SEQ ID NO:15, or SEQ ID NO:29; (b)an H-CDR2 as set forth in SEQ ID NO:2, SEQ ID NO:16, or SEQ ID NO:30;and (c) an H-CDR3 as set forth in SEQ ID NO:3, SEQ ID NO: 17, or SEQ IDNO:31. In a further aspect, such antibodies or antigen-binding portionsfurther comprise: (a) an L-CDR1 as set forth in SEQ ID NO:4, SEQ IDNO:18, or SEQ ID NO:32; (b) an L-CDR2 as set forth in SEQ ID NO:5, SEQID NO:19, or SEQ ID NO:33; and (c) an L-CDR3 as set forth in SEQ IDNO:6, SEQ ID NO:20, SEQ ID NO:34, or SEQ ID NO:75.

In a further aspect, the disclosure provides an isolated antibody, orantigen-binding portion thereof, that specifically binds osteopontin,wherein said antibody or antigen-binding portion comprises: (a) anL-CDR1 as set forth in SEQ ID NO:4, SEQ ID NO:18, or SEQ ID NO:32; (b)an L-CDR2 as set forth in SEQ ID NO:5, SEQ ID NO:19, or SEQ ID NO:33;and (c) an L-CDR3 as set forth in SEQ ID NO:6, SEQ ID NO:20, SEQ IDNO:34, or SEQ ID NO:75. In a further aspect, such antibodies orantigen-binding portions further comprise: (a) an H-CDR1 as set forth inSEQ ID NO:1, SEQ ID NO:15, or SEQ ID NO:29; (b) an H-CDR2 as set forthin SEQ ID NO:2, SEQ ID NO:16, or SEQ ID NO:30; and (c) an H-CDR3 as setforth in SEQ ID NO:3, SEQ ID NO:17, or SEQ ID NO:31.

In a further aspect, the disclosure provides an isolated antibody, orantigen-binding portion thereof, that specifically binds osteopontin,wherein said antibody or antigen-binding portion comprises an H-CDR1 asset forth in SEQ ID NO:1, an H-CDR2 as set forth in SEQ ID NO:2, and anH-CDR3 as set forth in SEQ ID NO:3.

In a further aspect, the disclosure provides an isolated antibody, orantigen-binding portion thereof, that specifically binds osteopontin,wherein said antibody or antigen-binding portion comprises an H-CDR1 asset forth in SEQ ID NO:15, an H-CDR2 as set forth in SEQ ID NO:16, andan H-CDR3 as set forth in SEQ ID NO:17.

In a further aspect, the disclosure provides an isolated antibody, orantigen-binding portion thereof, that specifically binds osteopontin,wherein said antibody or antigen-binding portion comprises an H-CDR1 asset forth in SEQ ID NO:29, an H-CDR2 as set forth in SEQ ID NO:30, andan H-CDR3 as set forth in SEQ ID NO:31.

In a further aspect, the disclosure provides an isolated antibody, orantigen-binding portion thereof, that specifically binds osteopontin,wherein said antibody or antigen-binding portion comprises an L-CDR1 asset forth in SEQ ID NO:4, an L-CDR2 as set forth in SEQ ID NO:5, and anL-CDR3 as set forth in SEQ ID NO:6.

In a further aspect, the disclosure provides an isolated antibody, orantigen-binding portion thereof, that specifically binds osteopontin,wherein said antibody or antigen-binding portion comprises an L-CDR1 asset forth in SEQ ID NO:18, an L-CDR2 as set forth in SEQ ID NO:19, andan L-CDR3 as set forth in SEQ ID NO:20.

In a further aspect, the disclosure provides an isolated antibody, orantigen-binding portion thereof, that specifically binds osteopontin,wherein said antibody or antigen-binding portion comprises an L-CDR1 asset forth in SEQ ID NO:32, an L-CDR2 as set forth in SEQ ID NO:33, andan L-CDR3 as set forth in SEQ ID NO:34.

In a further aspect, the disclosure provides an isolated antibody, orantigen-binding portion thereof, that specifically binds osteopontin,wherein said antibody or antigen-binding portion comprises an L-CDR1 asset forth in SEQ ID NO:4, an L-CDR2 as set forth in SEQ ID NO:5, and anL-CDR3 as set forth in SEQ ID NO:75.

In a further aspect, the disclosure provides an isolated antibody, orantigen-binding portion thereof, that specifically binds osteopontin,wherein said antibody or antigen-binding portion comprises an H-CDR1 asset forth in SEQ ID NO:1, an H-CDR2 as set forth in SEQ ID NO:2, anH-CDR3 as set forth in SEQ ID NO:3, an L-CDR1 as set forth in SEQ IDNO:4, an L-CDR2 as set forth in SEQ ID NO:5, and an L-CDR3 as set forthin SEQ ID NO:6.

In a further aspect, the disclosure provides an isolated antibody, orantigen-binding portion thereof, that specifically binds osteopontin,wherein said antibody or antigen-binding portion comprises an H-CDR1 asset forth in SEQ ID NO:15, an H-CDR2 as set forth in SEQ ID NO:16, anH-CDR3 as set forth in SEQ ID NO:17, an L-CDR1 as set forth in SEQ IDNO:18, an L-CDR2 as set forth in SEQ ID NO:19, and an L-CDR3 as setforth in SEQ ID NO:20.

In a further aspect, the disclosure provides an isolated antibody, orantigen-binding portion thereof, that specifically binds osteopontin,wherein said antibody or antigen-binding portion comprises an H-CDR1 asset forth in SEQ ID NO:29, an H-CDR2 as set forth in SEQ ID NO:30, anH-CDR3 as set forth in SEQ ID NO:31, an L-CDR1 as set forth in SEQ IDNO:32, an L-CDR2 as set forth in SEQ ID NO:33, and an L-CDR3 as setforth in SEQ ID NO:34.

In a further aspect, the disclosure provides an isolated antibody, orantigen-binding portion thereof, that specifically binds osteopontin,wherein said antibody or antigen-binding portion comprises an H-CDR1 asset forth in SEQ ID NO:1, an H-CDR2 as set forth in SEQ ID NO:2, anH-CDR3 as set forth in SEQ ID NO:3, an L-CDR1 as set forth in SEQ IDNO:4, an L-CDR2 as set forth in SEQ ID NO:5, and an L-CDR3 as set forthin SEQ ID NO:75.

In a further aspect, the disclosure provides an isolated antibody, orantigen-binding portion thereof, that specifically binds osteopontin,wherein said antibody or antigen-binding portion comprises a V_(H) chainamino acid sequence as set forth in SEQ ID NO:7, SEQ ID NO:21, SEQ IDNO:35, SEQ ID NO:44, SEQ ID NO:48, or SEQ ID NO:52. In one aspect, saidantibody or antigen-binding portion further comprises a V_(L) chainamino acid sequence as set forth in SEQ ID NO:8, SEQ ID NO:22, SEQ IDNO:36, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:56 or SEQ IDNO:76.

In a further aspect, the disclosure provides an isolated antibody orantigen-binding portion thereof that specifically binds osteopontin,wherein said antibody or antigen-binding portion comprises a V_(L) chainamino acid sequence as set forth in SEQ ID NO:8, SEQ ID NO:22, SEQ IDNO:36, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:56, or SEQ IDNO:76. In one embodiment, said antibody or antigen-binding portionfurther comprises a V_(H) chain amino acid sequence as set forth in SEQID NO:7, SEQ ID NO:21, SEQ ID NO:35, SEQ ID NO:44, SEQ ID NO:48, or SEQID NO:52.

In one aspect, the disclosure provides an isolated antibody, orantigen-binding portion thereof, that specifically binds osteopontin,wherein said antibody or antigen-binding portion comprises a V_(H) chainamino acid sequence as set forth in SEQ ID NO:7 and a V_(L) chain aminoacid sequence as set forth in SEQ ID NO:8.

In one aspect, the disclosure provides an isolated antibody, orantigen-binding portion thereof, that specifically binds osteopontin,wherein said antibody or antigen-binding portion comprises a V_(H) chainamino acid sequence as set forth in SEQ ID NO:21 and a V_(L) chain aminoacid sequence as set forth in SEQ ID NO:22.

In one aspect, the disclosure provides an isolated antibody, orantigen-binding portion thereof, that specifically binds osteopontin,wherein said antibody or antigen-binding portion comprises a V_(H) chainamino acid sequence as set forth in SEQ ID NO:35 and a V_(L) chain aminoacid sequence as set forth in SEQ ID NO:36.

In one aspect, the disclosure provides an isolated antibody, orantigen-binding portion thereof, that specifically binds osteopontin,wherein said antibody or antigen-binding portion comprises a V_(H) chainamino acid sequence as set forth in SEQ ID NO:44 and a V_(L) chain aminoacid sequence as set forth in SEQ ID NO:46.

In one aspect, the disclosure provides an isolated antibody, orantigen-binding portion thereof, that specifically binds osteopontin,wherein said antibody or antigen-binding portion comprises a V_(H) chainamino acid sequence as set forth in SEQ ID NO:48 and a V_(L) chain aminoacid sequence as set forth in SEQ ID NO:50.

In one aspect, the disclosure provides an isolated antibody, orantigen-binding portion thereof, that specifically binds osteopontin,wherein said antibody or antigen-binding portion comprises a V_(H) chainamino acid sequence as set forth in SEQ ID NO:52 and a V_(L) chain aminoacid sequence as set forth in SEQ ID NO:54.

In one aspect, the disclosure provides an isolated antibody, orantigen-binding portion thereof, that specifically binds osteopontin,wherein said antibody or antigen-binding portion comprises a V_(H) chainamino acid sequence as set forth in SEQ ID NO:52 and a V_(L) chain aminoacid sequence as set forth in SEQ ID NO:56.

In one aspect, the disclosure provides an isolated antibody, orantigen-binding portion thereof, that specifically binds osteopontin,wherein said antibody or antigen-binding portion comprises a V_(H) chainamino acid sequence as set forth in SEQ ID NO:7 and a V_(L) chain aminoacid sequence as set forth in SEQ ID NO:76.

In one aspect, the osteopontin to which any of the antibodies, orantigen-binding portions, described herein specifically bind is humanosteopontin. In a further aspect, said osteopontin is murineosteopontin.

In another aspect, the disclosure provides an antibody according to anyof the antibodies as described herein, which is an IgG. For example,said antibodies can be IgG1, IgG2, IgG3, or IgG4.

In a further aspect, the disclosure provides an antibody according toany of the antibodies as described herein, which is a human, humanized,or chimeric antibody.

In a further aspect, the disclosure provides an antigen-binding portionaccording to any of the antigen-binding portions described herein, whichis a Fab or scFv antibody fragment.

In one aspect, the C-terminal lysine of the heavy chain of any of theanti-OPN antibodies of the disclosure as described is cleaved, and isthus not present. For example, in a further aspect, the disclosureprovides an isolated antibody comprising a heavy chain amino acidsequence as set forth in SEQ ID NO:11; and a light chain amino acidsequence as set forth in SEQ ID NO:12, with the proviso that theC-terminal lysine residue of SEQ ID NO:11 is optionally not present.

In a further aspect, the disclosure provides an isolated antibodycomprising a heavy chain amino acid sequence as set forth in SEQ IDNO:25; and a light chain amino acid sequence as set forth in SEQ IDNO:26, with the proviso that the C-terminal lysine residue of SEQ IDNO:25 is optionally not present.

In a further aspect, the disclosure provides an isolated antibodycomprising a heavy chain amino acid sequence as set forth in SEQ IDNO:39; and a light chain amino acid sequence as set forth in SEQ IDNO:40, with the proviso that the C-terminal lysine residue of SEQ IDNO:39 is optionally not present.

In a further aspect, the disclosure provides an isolated antibodycomprising a heavy chain amino acid sequence as set forth in SEQ IDNO:58; and a light chain amino acid sequence as set forth in SEQ IDNO:59, with the proviso that the C-terminal lysine residue of SEQ IDNO:58 is optionally not present.

In a further aspect, the disclosure provides an isolated antibodycomprising a heavy chain amino acid sequence as set forth in SEQ IDNO:62; and a light chain amino acid sequence as set forth in SEQ IDNO:63, with the proviso that the C-terminal lysine residue of SEQ IDNO:62 is optionally not present.

In a further aspect, the disclosure provides an isolated antibodycomprising a heavy chain amino acid sequence as set forth in SEQ IDNO:66; and a light chain amino acid sequence as set forth in SEQ IDNO:67, with the proviso that the C-terminal lysine residue of SEQ IDNO:66 is optionally not present.

In a further aspect, the disclosure provides an isolated antibodycomprising a heavy chain amino acid sequence as set forth in SEQ IDNO:11; and a light chain amino acid sequence as set forth in SEQ IDNO:78, with the proviso that the C-terminal lysine residue of SEQ IDNO:11 is optionally not present.

In a still further aspect, the disclosure provides an isolated antibodyor antigen-binding portion thereof comprising a V_(H) chain that isencoded by (i) a nucleic acid sequence comprising SEQ ID NO:9, SEQ IDNO:23, SEQ ID NO:37, SEQ ID NO:45, SEQ ID NO:49, or SEQ ID NO:53, or(ii) a nucleic acid sequences that hybridizes under high stringencyconditions to the complementary strand of SEQ ID NO:9, SEQ ID NO:23, SEQID NO:37, SEQ ID NO:45, SEQ ID NO:49, or SEQ ID NO:53, wherein saidantibody or antigen-binding portion specifically binds osteopontin.

In a further aspect, the disclosure provides an isolated antibody, orantigen-binding portion thereof, that competes, and/or thatcross-competes for binding to OPN with any of the OPN antibodies orantigen-binding portions disclosed herein. For example, an antibody, orantigen binding portion thereof that specifically binds to OPN and thatcompetes for binding to OPN, and/or that cross-competes for binding toOPN with a monoclonal antibody selected from 6990, 6991, and 6993. Inone aspect, such isolated antibody is a human antibody. In anotheraspect, such isolated antibody is a humanized antibody.

In a further aspect there is provided an isolated antibody or antigenbinding portion thereof that binds to the same epitope on human OPN asany of the antibodies disclosed herein and/or competes for binding tohuman OPN with such an antibody. For example, an antibody, or antigenbinding portion thereof, that specifically binds to OPN, and that bindsto the same epitope on human OPN as a monoclonal antibody selected from6990, 6991, and 6993, and/or competes for binding to human OPN with amonoclonal antibody selected from 6990, 6991, and 6993.

A further aspect of the present disclosure is an isolated antibody, oran antigen-binding portion thereof, comprising a heavy chain variableregion that is the product of, or derived from, a human V_(H) 3-23 gene,wherein the antibody specifically binds OPN.

A further aspect of the present disclosure is an isolated monoclonalantibody, or an antigen-binding portion thereof, comprising a lightchain variable region that is the product of, or derived from, a humanV_(L) λ3 or λ1-13 gene, wherein the antibody specifically binds OPN.

In a further aspect, the disclosure provides an immunoconjugatecomprising any of the antibodies, or antigen-binding portions thereof,as described herein, linked to a therapeutic agent. In one case, thetherapeutic agent is a cytotoxin or a radioactive isotope. In a furtheraspect, the disclosure provides a composition comprising any of theimmunoconjugates described herein and a pharmaceutically acceptablecarrier. The disclosure also provides a bispecific molecule comprisingan antibody, or antigen-binding portion thereof, linked to a secondfunctional moiety having a different binding specificity than saidantibody, or antigen binding portion thereof.

In a further aspect, the disclosure provides a method for preparing ananti-OPN antibody comprising:

(a) providing: (i) a heavy chain variable region antibody sequencecomprising a H-CDR1 sequence selected from the group consisting of SEQID NOs: 1, 15, and 29, a H-CDR2 sequence selected from the groupconsisting of SEQ ID NOs: 2, 16, and 30, and/or a H-CDR3 sequenceselected from the group consisting of SEQ ID NOs: 3, 17, and 31; and/or(ii) a light chain variable region antibody sequence comprising a L-CDR1sequence selected from the group consisting of SEQ ID NOs: 4, 18, and32, a L-CDR2 sequence selected from the group consisting of SEQ ID NOs:5, 19, and 33, and/or a L-CDR3 sequence selected from the groupconsisting of SEQ ID NOs: 6, 20, 34, and 75; and

(b) expressing the antibody sequence as a protein.

In a further aspect, the disclosure provides a method for preparing ananti-OPN antibody comprising:

(a) providing: (i) a nucleic acid sequence that encodes a heavy chainvariable region antibody sequence comprising a H-CDR1 sequence selectedfrom the group consisting of SEQ ID NOs: 1, 15, and 29, a H-CDR2sequence selected from the group consisting of SEQ ID NOs: 2, 16, and30, and/or a H-CDR3 sequence selected from the group consisting of SEQID NOs: 3, 17, and 31; and/or (ii) a nucleic acid sequence that thatencodes a light chain variable region antibody sequence comprising aL-CDR1 sequence selected from the group consisting of SEQ ID NOs: 4, 18,and 32, a L-CDR2 sequence selected from the group consisting of SEQ IDNOs: 5, 19, and 33, and/or a L-CDR3 sequence selected from the groupconsisting of SEQ ID NOs: 6, 20, 34, and 75; and

(b) expressing the nucleic acid sequence to produce an antibody or anantigen-binding portion thereof.

In a further aspect, the disclosure provides a method for preparing ananti-OPN antibody comprising:

(a) providing: (i) a heavy chain variable region antibody sequencecomprising a H-CDR1 sequence selected from the group consisting of SEQID NOs: 1, 15, and 29, a H-CDR2 sequence selected from the groupconsisting of SEQ ID NOs: 2, 16, and 30, and/or a H-CDR3 sequenceselected from the group consisting of SEQ ID NOs: 3, 17, and 31; and/or(ii) a light chain variable region antibody sequence comprising a L-CDR1sequence selected from the group consisting of SEQ ID NOs: 4, 18, and32, a L-CDR2 sequence selected from the group consisting of SEQ ID NOs:5, 19, and 33, and/or a L-CDR3 sequence selected from the groupconsisting of SEQ ID NOs: 6, 20, 34, and 75;

(b) altering at least one amino acid residue within the heavy chainvariable region antibody sequence and/or the light chain variable regionantibody sequence to create at least one altered antibody sequence; and

(c) expressing the altered antibody sequence as a protein.

In a still further aspect, the disclosure provides an isolated antibodyor antigen-binding portion thereof comprising a V_(L) chain that isencoded by (i) a nucleic acid sequence comprising SEQ ID NO:10, SEQ IDNO:24, SEQ ID NO:38, SEQ ID NO:47, SEQ ID NO:51, SEQ ID NO:55, SEQ IDNO:57, or SEQ ID NO:77, or (ii) a nucleic acid sequences that hybridizesunder high stringency conditions to the complementary strand of SEQ IDNO:10, SEQ ID NO:24, SEQ ID NO:38, SEQ ID NO:47, SEQ ID NO:51, SEQ IDNO:55, SEQ ID NO:57, or SEQ ID NO:77, wherein said antibody orantigen-binding portion specifically binds OPN.

In a further aspect, the disclosure provides an isolated nucleic acidthat encodes any of the antibodies as described herein.

In a further aspect, the disclosure provides an isolated nucleic acidthat encodes a V_(H) chain of an antibody or antigen-binding portionthereof, and that comprises (i) SEQ ID NO:9, SEQ ID NO:23, SEQ ID NO:37,SEQ ID NO:45, SEQ ID NO:49, or SEQ ID NO:53; or (ii) a nucleic acidsequence that hybridizes under high stringency conditions to thecomplementary strand of SEQ ID NO:9, SEQ ID NO:23, SEQ ID NO:37, SEQ IDNO:45, SEQ ID NO:49, or SEQ ID NO:53; wherein said antibody orantigen-binding portion specifically binds OPN.

In a still further aspect is provided an isolated nucleic acid thatencodes a V_(L) chain of an antibody or antigen-binding portion thereof,and that comprises (i) SEQ ID NO:10, SEQ ID NO:24, SEQ ID NO:38, SEQ IDNO:47, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:57, or SEQ ID NO:77; or(ii) a nucleic acid sequence that hybridizes under high stringencyconditions to the complementary strand of SEQ ID NO:10, SEQ ID NO:24,SEQ ID NO:38, SEQ ID NO:47, SEQ ID NO:51, SEQ ID NO:55, SEQ ID NO:57, orSEQ ID NO:77; wherein said antibody or antigen-binding portionspecifically binds OPN.

In a further aspect, the disclosure provides a vector comprising any ofthe nucleic acids described herein. In a still further aspect, thedisclosure provides a host cell comprising any of the vectors describedherein. For example, such host cells can be bacterial or mammalian.

In a further aspect, the disclosure provides a pharmaceuticalcomposition comprising any of the antibodies or antigen-bindingportions, immunoconjugates, or bispecific molecules described herein anda pharmaceutically acceptable carrier or excipient.

The disclosure further provides methods for treating abnormal cellgrowth comprising administering to a subject in need thereof aneffective amount of any of the pharmaceutical compositions describedherein. The disclosure further provides methods of reducing tumor cellmetastasis in a subject, comprising administering to said subject aneffective amount of any of the antibodies, antigen-binding portions, orpharmaceutical compositions described herein.

In a further aspect, the disclosure provides a use of any of theantibodies, antigen-binding portions, or pharmaceutical compositionsdescribed herein, for the manufacture of a medicament for the treatmentof abnormal cell growth in a subject in need thereof. In a still furtheraspect, the disclosure provides a use of any of the antibodies,antigen-binding portions, or pharmaceutical compositions describedherein, for the manufacture of a medicament for the treatment of tumorcell metastasis in a subject in need thereof.

In a further aspect, the disclosure provides the antibodies,antigen-binding portions, or pharmaceutical compositions describedherein for use in the treatment of abnormal cell growth in a subject inneed thereof. In a still further aspect, the disclosure provides theantibodies, antigen-binding portions, or pharmaceutical compositionsdescribed herein for use in the treatment of tumor cell metastasis in asubject in need thereof.

In another aspect, the disclosure provides methods of preparing ananti-ostepontin antibody, or antigen-binding portion thereof, comprisingexpressing the antibody or antigen-binding portion in any of the hostcells described herein.

In a further aspect, the disclosure provides any of the humanantibodies, or antigen-binding portions thereof, wherein said humanantibodies or antigen-binding portions are synthetic human antibodies orantigen-binding portions.

The present disclosure further provides antibodies, or antigen-bindingportions thereof, comprising peptide variants of any of the specificsequences disclosed herein (e.g. SEQ ID NOs: 1 to 42, and 44 to 69).Such peptide variants can include both conservative and non-conservativesubstitutions, deletions, and/or additions, and typically includepeptides that are at least 60%, at least 65%, at least 70%, at least75%, at least 80%, at least 85%, at least 87%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% identicalto any of the specific sequences disclosed herein.

For example, in one aspect, the disclosure provides an isolated antibodyor antigen-binding portion thereof that comprises a V_(H) chain aminoacid sequence as set forth in SEQ ID NO:7, SEQ ID NO:21, SEQ ID NO:35,SEQ ID NO:44, SEQ ID NO:48, or SEQ ID NO:52 or a peptide variantthereof. In one aspect, said peptide variant comprises 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative or non-conservativesubstitutions, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or15 additions and/or deletions to SEQ ID NO:7, SEQ ID NO:21, SEQ IDNO:35, SEQ ID NO:44, SEQ ID NO:48, or SEQ ID NO:52. In a further aspect,said peptide variant is at least 65%, at least 75%, at least 85%, atleast 90%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% identical to SEQ ID NO:7, SEQ ID NO:21, SEQ ID NO:35, SEQ IDNO:44, SEQ ID NO:48, or SEQ ID NO:52, and wherein said antibody orantigen-binding portion specifically binds osteopontin.

In a further aspect, the disclosure provides an isolated antibody orantigen-binding portion thereof that comprises a V_(L) chain amino acidsequence as set forth in SEQ ID NO:8, SEQ ID NO:22, SEQ ID NO:36, SEQ IDNO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ ID NO:56, or SEQ ID NO:76 or apeptide variant thereof. In one aspect, said peptide variant comprises1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative ornon-conservative substitutions, and/or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, or 15 additions and/or deletions to SEQ ID NO:8, SEQ IDNO:22, SEQ ID NO:36, SEQ ID NO:46, SEQ ID NO:50, SEQ ID NO:54, SEQ IDNO:56, or SEQ ID NO:76. In a further aspect, said peptide variant is atleast 65%, at least 75%, at least 85%, at least 90%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% identical to SEQID NO:8, SEQ ID NO:22, SEQ ID NO:36, SEQ ID NO:46, SEQ ID NO:50, SEQ IDNO:54, SEQ ID NO:56, or SEQ ID NO:76, and wherein said antibody orantigen-binding portion specifically binds OPN.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows the DNA sequence of the MOR-6990 heavy chain variableregion—corresponding CDR regions are underlined (SEQ ID NO:9);

FIG. 1B shows the amino acid sequence of the MOR-6990 heavy chainvariable region (SEQ ID NO:7)—CDR regions are underlined;

FIG. 1C shows the DNA sequence of the MOR-6990 light chain variableregion—corresponding CDR regions are underlined (SEQ ID NO:10);

FIG. 1D shows the amino acid sequence of the MOR-6990 light chainvariable region (SEQ ID NO:8)—CDR regions are underlined;

FIG. 1E shows the DNA sequence of the MOR-6991 heavy chain variableregion—corresponding CDR regions are underlined (SEQ ID NO:23);

FIG. 1F shows the amino acid sequence of the MOR-6991 heavy chainvariable region (SEQ ID NO:21)—CDR regions are underlined;

FIG. 1G shows the DNA sequence of the MOR-6991 light chain variableregion—corresponding CDR regions are underlined (SEQ ID NO:24);

FIG. 1H shows the amino acid sequence of the MOR-6991 light chainvariable region (SEQ ID NO:20)—CDR regions are underlined;

FIG. 1I shows the DNA sequence of the MOR-6993 heavy chain variableregion—corresponding CDR regions are underlined (SEQ ID NO:37);

FIG. 1J shows the amino acid sequence of the MOR-6993 heavy chainvariable region (SEQ ID NO:35)—CDR regions are underlined;

FIG. 1K shows the DNA sequence of the MOR-6993 light chain variableregion—corresponding CDR regions are underlined (SEQ ID NO:38);

FIG. 1L shows the amino acid sequence of the MOR-6993 light chainvariable region (SEQ ID NO:36)—CDR regions are underlined;

FIG. 1M shows the DNA sequence of the MOR-6990-GL heavy chain variableregion, where germ line mutations are shown by boxing, and correspondingCDR regions are underlined (SEQ ID NO:45);

FIG. 1N shows the amino acid sequence of the MOR-6990-GL heavy chainvariable region, where germ line mutations are shown by boxing, andcorresponding CDR regions are underlined (SEQ ID NO:44);

FIG. 1O shows the DNA sequence of the MOR-6990-GL light chain variableregion, where germ line mutations are shown by boxing, and correspondingCDR regions are underlined (SEQ ID NO:47);

FIG. 1P shows the amino acid sequence of the MOR-6990-GL light chainvariable region, where germ line mutations are shown by boxing, andcorresponding CDR regions are underlined (SEQ ID NO:46);

FIG. 1Q shows the DNA sequence of the MOR-6991-GL heavy chain variableregion, where germ line mutations are shown by boxing, and correspondingCDR regions are underlined (SEQ ID NO:49);

FIG. 1R shows the amino acid sequence of the MOR-6991-GL heavy chainvariable region, where germ line mutations are shown by boxing, andcorresponding CDR regions are underlined (SEQ ID NO:48);

FIG. 1S shows the DNA sequence of the MOR-6991-GL light chain variableregion, where germ line mutations are shown by boxing, and correspondingCDR regions are underlined (SEQ ID NO:51);

FIG. 1T shows the amino acid sequence of the MOR-6991-GL light chainvariable region, where germ line mutations are shown by boxing, andcorresponding CDR regions are underlined (SEQ ID NO:50);

FIG. 1U shows the DNA sequence of the MOR-6993-GL heavy chain variableregion, where germ line mutations are shown by boxing, and correspondingCDR regions are underlined (SEQ ID NO:53);

FIG. 1V shows the amino acid sequence of the MOR-6993-GL heavy chainvariable region, where germ line mutations are shown by boxing, andcorresponding CDR regions are underlined (SEQ ID NO:52);

FIG. 1W shows the DNA sequence of the MOR-6993-GL light chain variableregion, where germ line mutations are shown by boxing, and correspondingCDR regions are underlined (SEQ ID NO:55);

FIG. 1X shows the amino acid sequence of the MOR-6993-GL light chainvariable region, where germ line mutations are shown by boxing, andcorresponding CDR regions are underlined (SEQ ID NO:54);

FIG. 1Y shows the DNA sequence of the MOR-6993-GL-V44K light chainvariable region, where germ line mutations are shown by boxing, the V44Kmutation is shown in bold, and corresponding CDR regions are underlined(SEQ ID NO:57);

FIG. 1Z shows the amino acid sequence of the MOR-6993-GL-V44K lightchain variable region, where germ line mutations are shown by boxing,the V44K is shown in bold, and corresponding CDR regions are underlined(SEQ ID NO:56);

FIG. 2A shows the DNA sequence of the MOR-6990 heavy chain (SEQ IDNO:13), where variable region sequence is shown in uppercase, whileconstant region sequence is shown in lowercase, and with correspondingCDR regions underlined;

FIG. 2B shows the amino acid sequence of the MOR-6990 heavy chain (SEQID NO:11), where variable region sequence is shown in uppercase, whileconstant region sequence is shown in lowercase, and with correspondingCDR regions underlined;

FIG. 2C shows the DNA sequence of the MOR-6990 light chain (SEQ IDNO:14), where variable region sequence is shown in uppercase, whileconstant region sequence is shown in lowercase, and with correspondingCDR regions underlined;

FIG. 2D shows the amino acid sequence of the MOR-6990 light chain (SEQID NO:12), where variable region sequence is shown in uppercase, whileconstant region sequence is shown in lowercase, and with correspondingCDR regions underlined;

FIG. 2E shows the DNA sequence of the MOR-6991 heavy chain (SEQ IDNO:27), where variable region sequence is shown in uppercase, whileconstant region sequence is shown in lowercase, and with correspondingCDR regions underlined;

FIG. 2F shows the amino acid sequence of the MOR-6991 heavy chain (SEQID NO:25), where variable region sequence is shown in uppercase, whileconstant region sequence is shown in lowercase, and with correspondingCDR regions underlined;

FIG. 2G shows the DNA sequence of the MOR-6991 light chain (SEQ IDNO:28), where variable region sequence is shown in uppercase, whileconstant region sequence is shown in lowercase, and with correspondingCDR regions underlined;

FIG. 2H shows the amino acid sequence of the MOR-6991 light chain (SEQID NO:26), where variable region sequence is shown in uppercase, whileconstant region sequence is shown in lowercase, and with correspondingCDR regions underlined;

FIG. 2I shows the DNA sequence of the MOR-6993 heavy chain (SEQ IDNO:41), where variable region sequence is shown in uppercase, whileconstant region sequence is shown in lowercase, and with correspondingCDR regions underlined;

FIG. 2J shows the amino acid sequence of the MOR-6993 heavy chain (SEQID NO:39), where variable region sequence is shown in uppercase, whileconstant region sequence is shown in lowercase, and with correspondingCDR regions underlined;

FIG. 2K shows the DNA sequence of the MOR-6993 light chain (SEQ IDNO:42), where variable region sequence is shown in uppercase, whileconstant region sequence is shown in lowercase, and with correspondingCDR regions underlined;

FIG. 2L shows the amino acid sequence of the MOR-6993 light chain (SEQID NO:40), where variable region sequence is shown in uppercase, whileconstant region sequence is shown in lowercase, and with correspondingCDR regions underlined;

FIG. 2M shows the DNA sequence of the MOR-6990-GL heavy chain (SEQ IDNO:60), where germ line mutations are shown by boxing, and correspondingCDR regions underlined; variable region sequence is shown in uppercase,while constant region sequence is lower case;

FIG. 2N shows the amino acid sequence of the MOR-6990-GL heavy chain(SEQ ID NO:58), where germ line mutations are shown by boxing, andcorresponding CDR regions underlined; variable region sequence is shownin uppercase, while constant region sequence is lower case;

FIG. 2O shows the DNA sequence of the MOR-6990-GL light chain (SEQ IDNO:61), where germ line mutations are shown by boxing, and correspondingCDR regions underlined; variable region sequence is shown in uppercase,while constant region sequence is lower case;

FIG. 2P shows the amino acid sequence of the MOR-6990-GL light chain(SEQ ID NO:59), where germ line mutations are shown by boxing, andcorresponding CDR regions underlined; variable region sequence is shownin uppercase, while constant region sequence is lower case;

FIG. 2Q shows the DNA sequence of the MOR-6991-GL heavy chain (SEQ IDNO:64), where germ line mutations are shown by boxing, and correspondingCDR regions underlined; variable region sequence is shown in uppercase,while constant region sequence is lower case;

FIG. 2R shows the amino acid sequence of the MOR-6991-GL heavy chain(SEQ ID NO:62), where germ line mutations are shown by boxing, andcorresponding CDR regions underlined; variable region sequence is shownin uppercase, while constant region sequence is lower case;

FIG. 2S shows the DNA sequence of the MOR-6991-GL light chain (SEQ IDNO:65), where germ line mutations are shown by boxing, and correspondingCDR regions underlined; variable region sequence is shown in uppercase,while constant region sequence is lower case;

FIG. 2T shows the amino acid sequence of the MOR-6991-GL light chain(SEQ ID NO:63), where germ line mutations are shown by boxing, andcorresponding CDR regions underlined; variable region sequence is shownin uppercase, while constant region sequence is lower case;

FIG. 2U shows the DNA sequence of the MOR-6993-GL heavy chain (SEQ IDNO:68), where germ line mutations are shown by boxing, and correspondingCDR regions underlined; variable region sequence is shown in uppercase,while constant region sequence is lower case;

FIG. 2V shows the amino acid sequence of the MOR-6993-GL heavy chain(SEQ ID NO:66), where germ line mutations are shown by boxing, andcorresponding CDR regions underlined; variable region sequence is shownin uppercase, while constant region sequence is lower case;

FIG. 2W shows the DNA sequence of the MOR-6993-GL light chain (SEQ IDNO:69), where germ line mutations are shown by boxing, and correspondingCDR regions underlined; variable region sequence is shown in uppercase,while constant region sequence is lower case;

FIG. 2X shows the amino acid sequence of the MOR-6993-GL light chain(SEQ ID NO:67), where germ line mutations are shown by boxing, andcorresponding CDR regions underlined; variable region sequence is shownin uppercase, while constant region sequence is lower case;

FIG. 3 shows the amino acid sequence of human OPN (SEQ ID NO:43),isoform b (Genbank NP_(—)000573).

FIG. 4A shows the amino acid sequence of the MOR-10475 light chainvariable region (SEQ ID NO:76)—CDR regions are underlined;

FIG. 4B shows the DNA sequence of the MOR-10475 light chain variableregion—corresponding CDR regions are underlined (SEQ ID NO:77);

FIG. 4C shows the amino acid sequence of the MOR-10475 light chain (SEQID NO:78), where variable region sequence is shown in uppercase, whileconstant region sequence is shown in lowercase, and with correspondingCDR regions underlined;

FIG. 5A shows the effect of MOR-6993 on tumor weight in a preclinicalmodel of breast cancer;

FIG. 5B shows the effect of MOR-6993 on metastasis in a preclinicalmodel of breast cancer;

FIG. 6A shows the neutralization of mouse osteopontin by MOR-6990 andMOR-6993;

FIG. 6B shows the neutralization of human osteopontin by MOR-6990 andMOR-6993.

DETAILED DESCRIPTION

The present disclosure is based on the discovery of novel antibodiesthat have a high affinity for osteopontin and can deliver a therapeuticbenefit to a subject. The antibodies of the present disclosure, whichmay be human, or humanized, can be used in many contexts, which are morefully described herein.

In order that the present disclosure may be more readily understood,certain terms are first defined. Additional definitions are set forththroughout the detailed description.

Unless otherwise defined herein, scientific and technical terms used inconnection with the present disclosure shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. Generally,nomenclatures used in connection with, and techniques of, cell andtissue culture, molecular biology, immunology, microbiology, geneticsand protein and nucleic acid chemistry and hybridization describedherein are those well known and commonly used in the art.

The methods and techniques of the present disclosure are generallyperformed according to methods well known in the art and as described invarious general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. Such references include, e.g., Sambrook and Russell,Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, ColdSpring Harbor, N.Y. (2001), Ausubel et al., Current Protocols inMolecular Biology, John Wiley & Sons, NY (2002), and Harlow and LaneAntibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y. (1990). Enzymatic reactions and purificationtechniques are performed according to manufacturer's specifications, ascommonly accomplished in the art or as described herein. Thenomenclatures used in connection with, and the laboratory procedures andtechniques of, analytical chemistry, synthetic organic chemistry, andmedicinal and pharmaceutical chemistry described herein are those wellknown and commonly used in the art. Standard techniques are used forchemical syntheses, chemical analyses, pharmaceutical preparation,formulation, and delivery, and treatment of patients.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage. See Immunology—A Synthesis (2ndEdition, E. S. Golub and D. R. Gren, Eds., Sinauer Associates,Sunderland, Mass. (1991)).

The term “antibody” as referred to herein includes whole antibodies andany antigen binding fragment (i.e., “antigen-binding portion”) or singlechains thereof. An “antibody” refers to a glycoprotein comprising atleast two heavy (H) chains and two light (L) chains inter-connected bydisulfide bonds, or an antigen binding portion thereof. Each heavy chainis comprised of a heavy chain variable region (abbreviated herein asV_(H)) and a heavy chain constant region. The heavy chain constantregion is comprised of three domains, C_(H1), C_(H2) and C_(H3). Eachlight chain is comprised of a light chain variable region (abbreviatedherein as V_(L)) and a light chain constant region. The light chainconstant region is comprised of one domain, C_(L). The V_(H) and V_(L)regions can be further subdivided into regions of hypervariability,termed complementarity determining regions (CDR), interspersed withregions that are more conserved, termed framework regions (FR). The CDRregions can be determined using the Kabat or Chothia numbering systems,both of which are well known to those of skill in the art. See, e.g.Kabat, E. A., et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242; Chothia and Lesk, J. Mol. Biol. 196:901-917(1987). Each V_(H) and V_(L) is composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. Throughout the present disclosure,the three CDRs of the heavy chain are referred to as H-CDR1, H-CDR2, andH-CDR3. Similarly, the three CDRs of the light chain are referred to asL-CDR1, L-CDR2, and L-CDR3. The variable regions of the heavy and lightchains contain a binding domain that interacts with an antigen. Theconstant regions of the antibodies may mediate the binding of theimmunoglobulin to host tissues or factors, including various cells ofthe immune system (e.g., effector cells) and the first component (Clq)of the classical complement system. Within light and heavy chains, thevariable and constant regions are joined by a “J” region of about 12 ormore amino acids, with the heavy chain also including a “D” region ofabout 10 or more amino acids. See generally, Fundamental Immunology Ch.7 (Paul, W., ed., 2^(nd) ed. Raven Press, N.Y. (1989)).

A “human” antibody, or antigen-binding portion thereof, is herebydefined as one that is not chimeric (e.g., not “humanized”) and not from(either in whole or in part) a non-human species. A human antibody orantigen-binding portion can be derived from a human or can be asynthetic human antibody. A “synthetic human antibody” is defined hereinas an antibody having a sequence derived, in whole or in part, in silicofrom synthetic sequences that are based on the analysis of known humanantibody sequences. In silico design of a human antibody sequence orfragment thereof can be achieved, for example, by analyzing a databaseof human antibody or antibody fragment sequences and devising apolypeptide sequence utilizing the data obtained therefrom. Anotherexample of a human antibody or antigen-binding portion, is one that isencoded by a nucleic acid isolated from a library of antibody sequencesof human origin (i.e., such library being based on antibodies taken froma human natural source).

A “humanized antibody”, or antigen-binding portion thereof, is definedherein as one that is (i) derived from a non-human source (e.g., atransgenic mouse which bears a heterologous immune system), whichantibody is based on a human germline sequence; or (ii) chimeric,wherein the variable domain is derived from a non-human origin and theconstant domain is derived from a human origin or (iii) CDR-grafted,wherein the CDRs of the variable domain are from a non-human origin,while one or more frameworks of the variable domain are of human originand the constant domain (if any) is of human origin. In the case wherethe CDRs are grafted from a non-human origin, said CDRs can besubsequently altered in order to improve the binding affinity to thetarget of interest.

As used herein, an antibody “specifically binds”, “specifically bindsto,” is “specific to/for” or “specifically recognizes” an antigen (here,osteopontin) if such antibody is able to discriminate between suchantigen and one or more reference antigen(s), since binding specificityis not an absolute, but a relative property. In its most general form(and when no defined reference is mentioned), “specific binding” isreferring to the ability of the antibody to discriminate between theantigen of interest and an unrelated antigen, as determined, forexample, in accordance with one of the following methods. Such methodscomprise, but are not limited to Western blots, ELISA-, RIA-, ECL-,IRMA-tests and peptide scans. For example, a standard ELISA assay can becarried out. The scoring may be carried out by standard colordevelopment (e.g. secondary antibody with horseradish peroxide andtetramethyl benzidine with hydrogenperoxide). The reaction in certainwells is scored by the optical density, for example, at 450 nm. Typicalbackground (=negative reaction) may be 0.1 OD; typical positive reactionmay be 1 OD. This means the difference positive/negative can be morethan 10-fold. Typically, determination of binding specificity isperformed by using not a single reference antigen, but a set of aboutthree to five unrelated antigens, such as milk powder, BSA, transferrinor the like. As used above, corresponding antigens from differentspecies are considered “related”, and are thus not “unrelated”. Forexample, unless indicated otherwise, an antibody or antigen-bindingportion described herein that binds both murine and human OPN isconsidered to “bind specifically” to OPN, provided that such antibody orantigen-binding portion does not also bind antigens that are“unrelated”, as described above. Typically, a specific or selectivereaction will be at least twice the background signal or noise and moretypically more than 10 times the background, even more specifically, anantibody is said to “specifically bind” an antigen when the equilibriumdissociation constant (K_(D)) is ≦1 μM, for example ≦100 nM and, furtherfor example, ≦10 nM.

The term “k_(on)”, as used herein, is intended to refer to the on-rate,or association rate of a particular antibody-antigen interaction,whereas the term “k_(off),” as used herein, is intended to refer to theoff-rate, or dissociation rate of a particular antibody-antigeninteraction. The term “K_(D)”, as used herein, is intended to refer tothe dissociation constant, which is obtained from the ratio of k_(off)to k_(on) (i.e., k_(off)/k_(on)) and is expressed as a molarconcentration (M). K_(D) values for antibodies can be determined usingmethods well established in the art. One method for determining theK_(D) of an antibody is by using surface plasmon resonance, typicallyusing a biosensor system such as a Biacore® system.

The term “compete”, as used herein with regard to an antibody, refers towhen a first antibody, or an antigen-binding portion thereof, competesfor binding with a second antibody, or an antigen-binding portionthereof, where binding of the first antibody with its cognate epitope isdetectably decreased in the presence of the second antibody compared tothe binding of the first antibody in the absence of the second antibody.The alternative, where the binding of the second antibody to its epitopeis also detectably decreased in the presence of the first antibody, can,but need not be the case. That is, a first antibody can inhibit thebinding of a second antibody to its epitope without that second antibodyinhibiting the binding of the first antibody to its respective epitope.However, where each antibody detectably inhibits the binding of theother antibody with its cognate epitope or ligand, whether to the same,greater, or lesser extent, the antibodies are said to “cross-compete”with each other for binding of their respective epitope(s). Forinstance, cross-competing antibodies can bind to the epitope, or portionof the epitope, to which the antibodies as disclosed herein bind. Use ofboth competing and cross-competing antibodies is encompassed by thepresent disclosure. Regardless of the mechanism by which suchcompetition or cross-competition occurs (e.g., steric hindrance,conformational change, or binding to a common epitope, or portionthereof, and the like), the skilled artisan would appreciate, based uponthe teachings provided herein, that such competing and/orcross-competing antibodies are encompassed and can be useful for themethods disclosed herein.

Also, as used herein, an “immunoglobulin” (Ig) is defined as a proteinbelonging to the class, or isotype, IgG, IgM, IgE, IgA, or IgD (or anysubclass thereof), and includes all conventionally known antibodies andantigen-binding portions thereof.

As used herein, “isotype” or “class” refers to the antibody class (e.g.,IgM or IgG) that is encoded by the heavy chain constant region genes.The constant domains of antibodies are not involved in binding toantigen, but exhibit various effector functions. Depending on the aminoacid sequence of the heavy chain constant region, a given human antibodyor immunoglobulin can be assigned to one of five major classes ofimmunoglobulins: IgA, IgD, IgE, IgG, and IgM. The structures andthree-dimensional configurations of different classes of immunoglobulinsare well-known. Of the various human immunoglobulin classes, only humanIgG1, IgG2, IgG3, IgG4, and IgM are known to activate complement. HumanIgG1 and IgG3 are known to mediate ADCC in humans.

As used herein, “subclass” refers to the further specification within anisotype of the heavy chain constant region gene, such as, for example,the IgG1, IgG2, IgG3, or IgG4 subclasses within the IgG isotype.

The term “epitope” includes any protein determinant capable of specificbinding to an immunoglobulin or T-cell receptor. Epitopic determinantsusually consist of chemically active surface groupings of molecules suchas amino acids or sugar side chains and usually have specific threedimensional structural characteristics, as well as specific chargecharacteristics. Conformational and nonconformational epitopes aredistinguished in that the binding to the former, but not the latter, islost in the presence of denaturing solvents.

The term “antigen-binding portion” of an antibody (or simply “antibodyportion”), as used herein, refers to one or more fragments of anantibody that retain the ability to specifically bind to an antigen(e.g., OPN). It has been shown that the antigen-binding function of anantibody can be performed by fragments of a full-length antibody.Examples of binding fragments encompassed within the term“antigen-binding portion” of an antibody include (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L) and C_(H1)domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and C_(H1) domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb fragment (Ward et al., Nature 341:544-546 (1989)),which consists of a V_(H) domain; and (vi) an isolated complementaritydetermining region (CDR). Furthermore, although the two domains of theFv fragment, V_(L) and V_(H), are coded for by separate genes, they canbe joined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the V_(L) and V_(H)regions pair to form monovalent molecules (known as single chain Fv(scFv). Such single chain antibodies are also intended to be encompassedwithin the term “antigen-binding portion” of an antibody. These antibodyfragments may be obtained using any suitable technique, includingconventional techniques known to those with skill in the art, and thefragments may be screened for utility in the same manner as are intactantibodies.

An “isolated antibody”, as used herein, is intended to refer to anantibody that is substantially free of other antibodies having differentantigenic specificities (e.g., an isolated antibody that specificallybinds OPN is substantially free of antibodies that specifically bindantigens other than OPN). An isolated antibody that specifically bindsOPN may, however, have cross-reactivity to other antigens, such as OPNmolecules from other species. Moreover, an isolated antibody may besubstantially free of other cellular material and/or chemicals.

The terms “monoclonal antibody” or “monoclonal antibody composition” asused herein refer to a preparation of antibody molecules of singlemolecular composition. A monoclonal antibody composition displays asingle binding specificity and affinity for a particular epitope.

The term “recombinant human antibody”, as used herein, includes allhuman antibodies that are prepared, expressed, created or isolated byrecombinant means, such as (a) antibodies isolated from an animal (e.g.,a mouse) that is transgenic or transchromosomal for human immunoglobulingenes or a hybridoma prepared therefrom (described further below), (b)antibodies isolated from a host cell transformed to express the humanantibody, e.g., from a transfectoma, (c) antibodies isolated from arecombinant, combinatorial human antibody library, and (d) antibodiesprepared, expressed, created or isolated by any other means that involvesplicing of human immunoglobulin gene sequences to other DNA sequences.Such recombinant human antibodies have variable regions in which theframework and CDR regions are derived from human germline immunoglobulinsequences. In certain embodiments, however, such recombinant humanantibodies can be subjected to in vitro mutagenesis (or, when an animaltransgenic for human Ig sequences is used, in vivo somatic mutagenesis)and thus the amino acid sequences of the V_(H) and V_(L) regions of therecombinant antibodies are sequences that, while derived from andrelated to human germline V_(H) and V_(L) sequences, may not naturallyexist within the human antibody germline repertoire in vivo.

As used herein, “sequence identity” between two polypeptide sequencesindicates the percentage of amino acids that are identical between thesequences. The amino acid sequence identity of polypeptides can bedetermined conventionally using known computer programs such as Bestfit,FASTA, or BLAST (see, e.g. Pearson, Methods Enzymol. 183:63-98 (1990);Pearson, Methods Mol. Biol. 132:185-219 (2000); Altschul et al., J. Mol.Biol. 215:403-410 (1990); Altschul et al., Nucelic Acids Res.25:3389-3402 (1997)). When using Bestfit or any other sequence alignmentprogram to determine whether a particular sequence is, for instance, 95%identical to a reference amino acid sequence, the parameters are setsuch that the percentage of identity is calculated over the full lengthof the reference amino acid sequence and that gaps in homology of up to5% of the total number of amino acid residues in the reference sequenceare allowed. This aforementioned method in determining the percentage ofidentity between polypeptides is applicable to all proteins, fragments,or variants thereof disclosed herein.

“Glycoform” refers to a complex oligosaccharide structure comprisinglinkages of various carbohydrate units. Such structures are describedin, e.g., Essentials of Glycobiology Varki et al., eds., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1999), which alsoprovides a review of standard glycobiology nomenclature. Such glycoformsinclude, but are not limited to, G2, G1, G0, G-1, and G-2 (see, e.g.,International Patent Publication No. WO 99/22764).

“Glycosylation pattern” is defined as the pattern of carbohydrate unitsthat are covalently attached to a protein (e.g., the glycoform) as wellas to the site(s) to which the glycoform(s) are covalently attached tothe peptide backbone of a protein, more specifically to animmunoglobulin protein. It is likely that antibodies expressed bydifferent cell lines or in transgenic animals will have differentglycoforms and/or glycosylation patterns compared with each other.However, all antibodies encoded by the nucleic acid molecules providedherein, or comprising the amino acid sequences provided herein are partof the present disclosure, regardless of the glycosylation of suchantibodies.

As used herein, the term “subject” includes any human or nonhumananimal. The term “nonhuman animal” includes all vertebrates, e.g.,mammals and non-mammals, such as nonhuman primates, sheep, dogs, cats,horses, cows, chickens, amphibians, reptiles, etc.

As used herein, the terms “treat”, “treating”, or “treatment”, withreference to a certain disease condition, mean reducing the frequencywith which symptoms of the disease (i.e., tumor growth and/ormetastasis, or other effect mediated by the numbers and/or activity ofimmune cells, and the like) are experienced by a subject. These termsinclude the administration of the compounds or agents of the presentdisclosure to prevent or delay the onset of the symptoms, complications,or biochemical indicia of the disease, or to alleviate the symptoms orarrest or inhibit further development of the disease, condition, ordisorder. Treatment may be prophylactic (to prevent or delay the onsetof the disease, or to prevent the manifestation of clinical orsubclinical symptoms thereof) or therapeutic suppression or alleviationof symptoms after the manifestation of the disease.

As used herein, the term “compound” or “pharmaceutical compound”includes antibodies, antigen-binding portions thereof, immunoconjugates,and bispecific molecules.

Antibodies of the Disclosure

Antibodies of the disclosure may be derived from a recombinant antibodylibrary that is based on amino acid sequences that have been designed insilico and encoded by nucleic acids that are synthetically created. Insilico design of an antibody sequence is achieved, for example, byanalyzing a database of human sequences and devising a polypeptidesequence utilizing the data obtained therefrom. Methods for designingand obtaining in silico-created sequences are described, for example, inKnappik et al., J. Mol. Biol. (2000) 296:57; Krebs et al., J. Immunol.Methods. (2001) 254:67; and U.S. Pat. No. 6,300,064 issued to Knappik etal.

Throughout this disclosure, reference is made to the followingrepresentative antibodies of the disclosure: MOR-6990 (6990), MOR-6991(6991), MOR-6993 (6993), and MOR-10475 (10475). As further described inExample 5, 6990 represents an antibody having a variable heavy regioncorresponding to SEQ ID NO:7, and a variable light region correspondingto SEQ ID NO:8; 6991 represents an antibody having a variable heavyregion corresponding to SEQ ID NO:21, and a variable light regioncorresponding to SEQ ID NO:22; and 6993 represents an antibody having avariable heavy region corresponding to SEQ ID NO:35, and a variablelight region corresponding to SEQ ID NO:36. 10475 represents an antibodyhaving a variable heavy region corresponding to SEQ ID NO:7, and avariable light region corresponding to SEQ ID NO:76.

The amino acid CDR sequences for the 6990, 6991, 6993, and 10475representative antibodies are shown below in Table 1.

TABLE 1 CDR sequences of antibodies 6990, 6991, and 6993. MOR-6990H-CDR1: SNYVMH (SEQ ID NO: 1) H-CDR2: SIFGSGSDTYYADSVKG (SEQ ID NO: 2)H-CDR3: RSASSGFGFAGYGIDS (SEQ ID NO: 3) L-CDR1: SGDSLRYYYAH(SEQ ID NO: 4) L-CDR2: DDNKRPS (SEQ ID NO: 5) L-CDR3: QSWDLFHSSV(SEQ ID NO: 6) MOR-6991 H-CDR1: NNYAVS (SEQ ID NO: 15)H-CDR2: GISYGGSNTYYADSVKG (SEQ ID NO: 16) H-CDR3: RTLGGDFDH(SEQ ID NO: 17) L-CDR1: SGSSSNIGSNYVN (SEQ ID NO: 18) L-CDR2: GNSKRPS(SEQ ID NO: 19) L-CDR3: QSFTQMLLV (SEQ ID NO: 20) MOR-6993H-CDR1: TTSSMH (SEQ ID NO: 29) H-CDR2: RISSHGSNTYYADSVKG (SEQ ID NO: 30)H-CDR3: RDMYRGVYGFAL (SEQ ID NO: 31) L-CDR1: SGDAIRNYYVH (SEQ ID NO: 32)L-CDR2: EDSDRPS (SEQ ID NO: 33) L-CDR3: QSYDKSNVV (SEQ ID NO: 34)MOR-10475 H-CDR1: SNYVMH (SEQ ID NO: 1) H-CDR2: SIFGSGSDTYYADSVKG(SEQ ID NO: 2) H-CDR3: RSASSGFGFAGYGIDS (SEQ ID NO: 3)L-CDR1: SGDSLRYYYAH (SEQ ID NO: 4) L-CDR2: DDNKRPS (SEQ ID NO: 5)L-CDR3: QAWDLINSHV (SEQ ID NO: 75)

In one aspect, the disclosure provides antibodies having anantigen-binding region that can bind specifically to or has a highaffinity for, one or more regions of human osteopontin, whose amino acidsequence is set forth in SEQ ID NO:43, and in FIG. 3. An antibody issaid to have a “high affinity” for an antigen if the affinitymeasurement is at least 100 nM (monovalent affinity of Fab fragment). Anantibody or antigen-binding portion of the present disclosure typicallybinds to human osteopontin with an affinity of about less than 500 nM,for example less than about 100 nM, less than about 60 nM, less thanabout 30 nM, less than about 10 nM, or less than about 3 nM. Exemplaryantibodies of the present disclosure and their corresponding bindingaffinities for osteopontin are further described in Examples 2 and 3herein.

The present disclosure also provides CDR portions of antibodies toosteopontin (including Chothia and Kabat CDRs). Determination of CDRregions is well within the skill of the art. It is understood that insome embodiments, CDRs can be a combination of the Kabat and Chothia CDR(also termed “combined CDRs” or “extended CDRs”). In some embodiments,the CDRs are the Kabat CDRs. In other embodiments, the CDRs are theChothia CDRs. In other words, in embodiments with more than one CDR, theCDRs may be any of Kabat, Chothia, combination CDRs, or combinationsthereof.

An antibody of the present disclosure can be species cross-reactive withhumans and at least one other species, which may be murine or rat. Anantibody that is cross reactive with at least one osteopontin species,for example, can provide greater flexibility and benefits over knownanti-osteopontin antibodies, for purposes of conducting in vivo studiesin multiple species with the same antibody.

Preferably, an antibody of the disclosure not only is able to bind toOPN, but also is able to reduce tumor cell metastasis and/or reduceabnormal cell growth, such as cancer.

Antibodies Having Particular Germline Sequences

In certain aspects, an antibody of the disclosure comprises a heavychain variable region from a particular germline heavy chainimmunoglobulin gene and/or a light chain variable region from aparticular germline light chain immunoglobulin gene.

For example, in one aspect, the disclosure provides an isolatedmonoclonal antibody, or an antigen-binding portion thereof, comprising aheavy chain variable region that is the product of, or derived from, ahuman V_(H) 3-23 gene, wherein the antibody specifically binds OPN. Inyet another aspect, the disclosure provides an isolated monoclonalantibody, or an antigen-binding portion thereof, comprising a lightchain variable region that is the product of, or derived from, a humanV_(L) λ3, or λ1-13 gene, wherein the antibody specifically binds OPN. Inyet another illustrative aspect, the disclosure provides an isolatedmonoclonal antibody, or antigen-binding portion thereof, wherein theantibody:

(a) comprises a heavy chain variable region that is the product of, orderived from, a human V_(H) 3-23 gene (which gene encodes the amino acidsequences set forth in SEQ ID NOs: 7, 21, and 35);

(b) comprises a light chain variable region that is the product of, orderived from, a human V_(L) λ3, or λ1-13 gene (which genes encodes theamino acid sequences set forth in SEQ ID NOs: 8, 36, or 22,respectively); and

(c) specifically binds to OPN, preferably human OPN.

Examples of antibodies having V_(H) and V_(L) of V_(H) 3-23 and V_(L)λ3, respectively, are 6990 and 6993. An example of an antibody havingV_(H) and V_(L) of V_(H) 3-23 and V_(L) λ1-13, respectively, is 6991.

As used herein, a human antibody comprises heavy or light chain variableregions that is “the product of” or “derived from” a particular germlinesequence if the variable regions of the antibody are obtained from asystem that uses human germline immunoglobulin genes. Such systemsinclude immunizing a transgenic mouse carrying human immunoglobulingenes with the antigen of interest or screening a human immunoglobulingene library displayed on phage with the antigen of interest. A humanantibody that is “the product of” or “derived from” a human germlineimmunoglobulin sequence can be identified as such by comparing the aminoacid sequence of the human antibody to the amino acid sequences of humangermline immunoglobulins and selecting the human germline immunoglobulinsequence that is closest in sequence (i.e., greatest % identity) to thesequence of the human antibody. A human antibody that is “the productof” or “derived from” a particular human germline immunoglobulinsequence may contain amino acid differences as compared to the germlinesequence, due to, for example, naturally-occurring somatic mutations orintentional introduction of site-directed mutation. However, a selectedhuman antibody typically is at least 90% identical in amino acidsequence to an amino acid sequence encoded by a human germlineimmunoglobulin gene and contains amino acid residues that identify thehuman antibody as being human when compared to the germlineimmunoglobulin amino acid sequences of other species (e.g., murinegermline sequences). In certain cases, a human antibody may be at least95%, or even at least 96%, 97%, 98%, or 99% identical in amino acidsequence to the amino acid sequence encoded by the germlineimmunoglobulin gene. In certain cases, the human antibody is identicalin amino acid sequence to the amino acid sequence encoded by thegermline Ig gene. Typically, a human antibody derived from a particularhuman germline sequence will display no more than 10 amino aciddifferences from the amino acid sequence encoded by the human germlineimmunoglobulin gene. In certain cases, the human antibody may display nomore than 5, or even no more than 4, 3, 2, or 1 amino acid differencesfrom the amino acid sequence encoded by the germline immunoglobulingene.

Antibodies that Bind the Same Epitope as the Antibodies of theDisclosure

In another aspect, the disclosure provides antibodies that bind to thesame epitope on human OPN as any of the illustrative OPN monoclonalantibodies of the disclosure (i.e., antibodies that have the ability tocross-compete for binding to OPN with any of the monoclonal antibodiesof the disclosure). For example, the reference antibody forcross-competition studies can be the monoclonal antibody 6990 (havingV_(H) and V_(L) sequences as shown in SEQ ID NOs: 7 and 8,respectively), or the monoclonal antibody 6991 (having V_(H) and V_(L)sequences as shown in SEQ ID NOs: 21 and 22, respectively), or themonoclonal antibody 6993 (having V_(H) and V_(L) sequences as shown inSEQ ID NOs: 35 and 36, respectively). Such cross-competing antibodiescan be identified based on their ability to cross-compete with 6990,6991, or 6993 in standard OPN binding assays. For example, BIAcoreanalysis, ELISA assays or flow cytometry may be used to demonstratecross-competition with the illustrative antibodies of the currentdisclosure. The ability of a test antibody to inhibit the binding of,for example, 6990, 6991, or 6993 to human OPN demonstrates that the testantibody can compete with 6990, 6991, or 6993 for binding to human OPNand thus binds to the same epitope on human OPN as 6990, 6991, or 6993.In one case, the antibody that binds to the same epitope on human OPN as6990, 6991, or 6993 is a human monoclonal antibody. Such humanmonoclonal antibodies can be prepared and isolated as described, forexample, in the Examples.

Antibody Variants

Antibodies of the present disclosure are not limited to the specificpeptide sequences provided herein. Rather, the disclosure also providesvariants of these polypeptides. With reference to the instant disclosureand conventionally available technologies and references, the skilledworker will be able to prepare, test and utilize functional variants ofthe antibodies disclosed herein, while appreciating that variants havingthe ability to specifically bind to OPN fall within the scope of thepresent disclosure.

As used herein, the term “peptide variant” or “antibody variant”encompasses both conservative and non-conservative substitutions,additions, and deletions, and can include, for example, an antibody thathas at least one altered CDR (hypervariable) and/or framework (FR)(variable) domain/position, vis-à-vis a peptide sequence disclosedherein. For example, it is well known in the art that theantigen-binding site of an antibody is formed by one or more CDRs, yetthe FR regions provide the structural framework for the CDRs and, hence,play an important role in antigen binding. By altering one or more aminoacid residues in a CDR or FR region, the skilled worker routinely cangenerate mutated or diversified antibody sequences, which can bescreened against the antigen, for new or improved properties, forexample.

FIGS. 1 and 2 show the VH and VL sequences for certain antibodies of thepresent disclosure, where the CDR regions are indicated by underline.The skilled worker can use the sequence information described herein todesign peptide variants that are within the scope of the presentdisclosure. For example, variants can be constructed by changing aminoacids within one or more CDR regions; a variant might also have one ormore altered framework regions. For example, a peptide FR domain mightbe altered where there is a deviation in a residue compared to agermline sequence.

To determine which amino acid residues to modify, the skilled worker cancompare the amino acid sequences disclosed herein to known sequences ofthe same class of such antibodies, using, for example, the proceduredescribed by Knappik et al., J. Mol. Biol. 296:57 (2000) and U.S. Pat.No. 6,300,064.

For example, variants may be obtained by diversifying one or more aminoacid residues in one or more CDRs, and by screening the resultingcollection of antibody variants for variants with improved properties.Particularly preferred is diversification of one or more amino acidresidues in L-CDR3, H-CDR3, L-CDR1, and/or H-CDR2. Diversification canbe done by synthesizing a collection of DNA molecules usingtrinucleotide mutagenesis (TRIM) technology (Virnekas, et al., Nucl.Acids Res. 22:5600 (1994)). For example, MOR-10475 was obtained bydiversifying amino acids in L-CDR3 of MOR-6990.

Conservative Amino Acid Substitutions

Polypeptide variants may be made that conserve the overall molecularstructure of an antibody peptide sequence described herein. Given theproperties of the individual amino acids, some rational substitutionswill be recognized by the skilled worker. Conservative amino acidsubstitutions may be made, for instance, on the basis of similarity inpolarity, charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues involved.

For example, (a) nonpolar (hydrophobic) amino acids include alanine,leucine, isoleucine, valine, proline, phenylalanine, tryptophan, andmethionine; (b) polar neutral amino acids include glycine, serine,threonine, cysteine, tyrosine, asparagine, and glutamine; (c) positivelycharged (basic) amino acids include arginine, lysine, and histidine; and(d) negatively charged (acidic) amino acids include aspartic acid andglutamic acid. Substitutions typically may be made within groups(a)-(d). In addition, glycine and proline may be substituted for oneanother based on their ability to disrupt α-helices. Similarly, certainamino acids, such as alanine, cysteine, leucine, methionine, glutamicacid, glutamine, histidine and lysine are more commonly found inα-helices, while valine, isoleucine, phenylalanine, tyrosine, tryptophanand threonine are more commonly found in β-pleated sheets. Glycine,serine, aspartic acid, asparagine, and proline are commonly found inturns. Some preferred substitutions may be made among the followinggroups: (i) S and T; (ii) P and G; and (iii) A, V, L and I. Given theknown genetic code, and recombinant and synthetic DNA techniques, theskilled scientist readily can construct DNAs encoding the conservativeamino acid variants.

Engineered and Modified Antibodies

An antibody, or antigen binding portion thereof, of the presentdisclosure can be prepared using an antibody having one or more of theV_(H) and/or V_(L) sequences disclosed herein as starting material toengineer a modified antibody, which modified antibody may have alteredproperties from the starting antibody. An antibody can be engineered bymodifying one or more residues within one or both variable regions(i.e., V_(H) and/or V_(L)), for example within one or more CDR regionsand/or within one or more framework regions. Additionally oralternatively, an antibody can be engineered by modifying residueswithin the constant region(s), for example to alter the effectorfunction(s) of the antibody.

One type of variable region engineering that can be performed is CDRgrafting. Antibodies interact with target antigens predominantly throughamino acid residues that are located in the six heavy and light chaincomplementarity determining regions (CDRs). For this reason, the aminoacid sequences within CDRs are more diverse between individualantibodies than sequences outside of CDRs. Because CDR sequences areresponsible for most antibody-antigen interactions, it is possible toexpress recombinant antibodies that mimic the properties of specificnaturally occurring antibodies by constructing expression vectors thatinclude CDR sequences from the specific naturally occurring antibodygrafted onto framework sequences from a different antibody withdifferent properties (see, e.g., Riechmann, L. et al. Nature 332:323-327(1998); Jones, P. et al. Nature 321:522-525 (1986); Queen, C. et al.Proc. Natl. Acad. Sci. U.S.A. 86:10029-10033 (1989); U.S. Pat. No.5,225,539; and U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,762 and6,180,370)

Accordingly, another aspect of the disclosure pertains to isolatedantibodies, or antigen binding portions thereof, that contain the V_(H)and V_(L) CDR sequences of the monoclonal antibodies 6990, 6991, and6993, yet may contain different framework sequences from theseantibodies.

Such framework sequences can be obtained from public DNA databases orpublished references that include germline antibody gene sequences. Forexample, germline DNA sequences for human heavy and light chain variableregion genes can be found in the “VBase” human germline sequencedatabase (Kabat, E. A., et al., Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242 (1991); Tomlinson, I. M., et al., J. Mol.Biol. 227:776-798 (1992); and Cox, J. P. L. et al., Eur. J. Immunol.24:827-836 (1994)). As another example, the germline DNA sequences forhuman heavy and light chain variable region genes can be found in theGenbank database.

Framework sequences for use in the antibodies of the disclosure include,but are not limited to, those that are structurally similar to theframework sequences used by selected antibodies of the disclosure, e.g.,similar to the V_(H) 3-23 framework sequences and/or the V_(L) λ3 orλ1-13 framework sequences used by illustrative antibodies of thedisclosure. For example, the H-CDR1, H-CDR2, and H-CDR3 sequences, andthe L-CDR1, L-CDR2, and L-CDR3 sequences, can be grafted onto frameworkregions that have the identical sequence as that found in the germlineimmunoglobulin gene from which the framework sequence derive, or the CDRsequences can be grafted onto framework regions that contain one or moremutations as compared to the germline sequences. For example, it hasbeen found that in certain instances it is beneficial to mutate residueswithin the framework regions to maintain or enhance the antigen bindingability of the antibody (see e.g., U.S. Pat. Nos. 5,530,101; 5,585,089;5,693,762 and 6,180,370).

Another type of variant is to mutate amino acid residues within theV_(H) and/or V_(L) CDR1, CDR2 and/or CDR3 regions to thereby improve oneor more binding properties (e.g., affinity) of the antibody of interest.Site-directed mutagenesis or PCR-mediated mutagenesis can be performedto introduce the mutation(s) and the effect on antibody binding, orother functional property of interest, can be evaluated in in vitro orin vivo assays as described herein and provided in the Examples.Typically, conservative substitutions (as discussed above) areintroduced. The mutations may be amino acid additions and/or deletions.Moreover, typically no more than one, two, three, four or five residueswithin a CDR region are altered.

Engineered antibodies of the disclosure include those in whichmodifications have been made to framework residues within the V_(H)and/or V_(L) regions, e.g., to improve the properties of the antibody.Typically such framework variants are made to decrease theimmunogenicity of the antibody. For example, one approach is to“backmutate” one or more framework residues to the correspondinggermline sequence. More specifically, an antibody that has undergonesomatic mutation may contain framework residues that differ from thegermline sequence from which the antibody is derived. Such residues canbe identified by comparing the antibody framework sequences to thegermline sequences from which the antibody is derived. To return theframework region sequences to their germline configuration, the somaticmutations can be “backmutated” to the germline sequence by, for example,site-directed mutagenesis or PCR-mediated mutagenesis. Several of theOPN antibodies of the present disclosure underwent such “back-mutations”to certain germline sequences, as described further in Example 6.

Another type of framework modification involves mutating one or moreresidues within the framework region, or even within one or more CDRregions, to remove T cell epitopes to thereby reduce the potentialimmunogenicity of the antibody. This approach is also referred to as“deimmunization” and is described in further detail in U.S. PatentPublication No. 2003-0153043.

To create an engineered antibody, it is not necessary to actuallyprepare (i.e., express as a protein) an antibody having one or more ofthe V_(H) and/or V_(L) sequences provided herein, or one or more CDRregions thereof. Rather, the information contained in the sequence(s)may be used as the starting material to create a “second generation”sequence(s) derived from the original sequence(s) and then the “secondgeneration” sequence(s) is prepared and expressed as a protein. Standardmolecular biology techniques can be used to prepare and express thealtered antibodies. Preferably, the altered antibody sequence(s) is onethat retains one, some or all of the functional properties of the OPNantibodies described herein. The functional properties of the alteredantibodies can be assessed using standard assays available in the artand/or described herein, such as those set forth in the Examples.

In certain aspects of the methods of engineering antibodies of thedisclosure, mutations can be introduced randomly or selectively alongall or part of an OPN antibody coding sequence and the resultingmodified OPN antibodies can be screened for binding activity and/orother functional properties as described herein. Mutational methods havebeen described in the art. For example, PCT Publication WO 02/092780describes methods for creating and screening antibody mutations usingsaturation mutagenesis, synthetic ligation assembly, or a combinationthereof. Alternatively, PCT Publication WO 03/074679 describes methodsof using computational screening methods to optimize physiochemicalproperties of antibodies.

In addition or alternative to modifications made within the framework orCDR regions, antibodies of the disclosure may be engineered to includemodifications within the Fc region, typically to alter one or morefunctional properties of the antibody, such as serum half-life,complement fixation, Fc receptor binding, and/or antigen-dependentcellular cytotoxicity. Furthermore, an antibody of the disclosure may bechemically modified (e.g., one or more chemical moieties can be attachedto the antibody) or be modified to alter its glycosylation pattern,again to alter one or more functional properties of the antibody. Eachof these aspects is described in further detail below. The numbering ofresidues in the Fc region is that of the EU index of Kabat.

In one case, the hinge region of CH1 is modified such that the number ofcysteine residues in the hinge region is altered, e.g., increased ordecreased. This approach is described further in U.S. Pat. No.5,677,425. The number of cysteine residues in the hinge region of CH1 isaltered to, for example, facilitate assembly of the light and heavychains or to increase or decrease the stability of the antibody.

In another case, the Fc hinge region of an antibody is mutated todecrease the biological half life of the antibody. More specifically,one or more amino acid mutations are introduced into the CH2-CH3 domaininterface region of the Fc-hinge fragment such that the antibody hasimpaired Staphylococcyl protein A (SpA) binding relative to nativeFc-hinge domain SpA binding. This approach is described in furtherdetail in U.S. Pat. No. 6,165,745.

In another case, the antibody is modified to increase its biologicalhalf life. Various approaches are possible. For example, one or more ofthe following mutations can be introduced: T252L, T254S, T256F, asdescribed in U.S. Pat. No. 6,277,375. Alternatively, to increase thebiological half life, the antibody can be altered within the CH1 or CLregion to contain a salvage receptor binding epitope taken from twoloops of a CH2 domain of an Fc region of an IgG, as described in U.S.Pat. Nos. 5,869,046 and 6,121,022.

In yet other cases, the Fc region is altered by replacing at least oneamino acid residue with a different amino acid residue to alter theeffector function(s) of the antibody. For example, one or more aminoacids selected from amino acid residues 234, 235, 236, 237, 297, 318,320 and 322 can be replaced with a different amino acid residue suchthat the antibody has an altered affinity for an effector ligand butretains the antigen-binding ability of the parent antibody. The effectorligand to which affinity is altered can be, for example, an Fc receptoror the C1 component of complement. This approach is described in furtherdetail in U.S. Pat. Nos. 5,624,821 and 5,648,260.

In another case, one or more amino acids selected from amino acidresidues 329, 331 and 322 can be replaced with a different amino acidresidue such that the antibody has altered C1q binding and/or reduced orabolished complement dependent cytotoxicity (CDC). This approach isdescribed in further detail in U.S. Pat. No. 6,194,551.

In another example, one or more amino acid residues within amino acidpositions 231 and 239 are altered to thereby alter the ability of theantibody to fix complement. This approach is described further in PCTPublication WO 94/29351.

In yet another example, the Fc region is modified to increase theability of the antibody to mediate antibody dependent cellularcytotoxicity (ADCC) and/or to increase the affinity of the antibody foran Fcγ receptor by modifying one or more amino acids at the followingpositions: 238, 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268,269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294,295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326,327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378,382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. Thisapproach is described further in PCT Publication WO 00/42072. Moreover,the binding sites on human IgG1 for FcγR1, FcγRII, FcγRIII and FcRn havebeen mapped and variants with improved binding have been described (seeShields et al., J. Biol. Chem. 276:6591-6604 (2001)). Specific mutationsat positions 256, 290, 298, 333, 334 and 339 were shown to improvebinding to FcγRIII. Additionally, the following combination mutants wereshown to improve FcγRIII binding: T256A/S298A, S298A/E333A, S298A/K224Aand S298A/E333A/K334A.

In still another example, the glycosylation of an antibody is modified.For example, an aglycoslated antibody can be made (i.e., the antibodylacks glycosylation). Glycosylation can be altered to, for example,increase the affinity of the antibody for antigen. Such carbohydratemodifications can be accomplished by, for example, altering one or moresites of glycosylation within the antibody sequence. For example, one ormore amino acid substitutions can be made that result in elimination ofone or more variable region framework glycosylation sites to therebyeliminate glycosylation at that site. Such aglycosylation may increasethe affinity of the antibody for antigen. Such an approach is describedin further detail in U.S. Pat. Nos. 5,714,350 and 6,350,861.

Additionally or alternatively, an antibody can be made that has analtered type of glycosylation, such as a hypofucosylated antibody havingreduced amounts of fucosyl residues or an antibody having increasedbisecting GlcNac structures. Such altered glycosylation patterns havebeen demonstrated to increase the ADCC ability of antibodies. Suchcarbohydrate modifications can be accomplished by, for example,expressing the antibody in a host cell with altered glycosylationmachinery. Cells with altered glycosylation machinery have beendescribed in the art and can be used as host cells in which to expressrecombinant antibodies of the disclosure to thereby produce an antibodywith altered glycosylation. For example, the cell lines Ms704, Ms705,and Ms709 lack the fucosyltransferase gene, FUT8 (alpha (1,6)fucosyltransferase), such that antibodies expressed in the Ms704, Ms705,and Ms709 cell lines lack fucose on their carbohydrates. The Ms704,Ms705, and Ms709 FUT8^(−/−) cell lines were created by the targeteddisruption of the FUT8 gene in CHO/DG44 cells using two replacementvectors (see U.S. Patent Publication No. 2004-0110704, and Yamane-Ohnukiet al., Biotechnol Bioeng 87:614-22 (2004)). As another example,European Patent Publication No. EP1,176,195 describes a cell line with afunctionally disrupted FUT8 gene, which encodes a fucosyl transferase,such that antibodies expressed in such a cell line exhibithypofucosylation by reducing or eliminating the alpha 1,6 bond-relatedenzyme. EP1,176,195 also describe cell lines which have a low enzymeactivity for adding fucose to the N-acetylglucosamine that binds to theFc region of the antibody or does not have the enzyme activity, forexample the rat myeloma cell line YB2/0 (ATCC CRL 1662). PCT PublicationWO 03/035835 describes a variant CHO cell line, Lec13 cells, withreduced ability to attach fucose to Asn(297)-linked carbohydrates, alsoresulting in hypofucosylation of antibodies expressed in that host cell(see also Shields et al., J. Biol. Chem. 277:26733-26740 (2002)). PCTPublication WO 99/54342 describes cell lines engineered to expressglycoprotein-modifying glycosyl transferases (e.g.,beta(1,4)-N-acetylglucosaminyltransferase III (GnTIII)) such thatantibodies expressed in the engineered cell lines exhibit increasedbisecting GlcNac structures which results in increased ADCC activity ofthe antibodies (see also Umana et al., Nat. Biotech. 17:176-180 (1999)).Alternatively, the fucose residues of the antibody may be cleaved offusing a fucosidase enzyme. For example, the fucosidasealpha-L-fucosidase removes fucosyl residues from antibodies (Tarentinoet al., (1975) Biochem. 14:5516-23 (1975)).

Another modification of the antibodies herein that is contemplated bythe disclosure is pegylation. An antibody can be pegylated to, forexample, increase the biological (e.g., serum) half life of theantibody. To pegylate an antibody, the antibody, or fragment thereof,typically is reacted with polyethylene glycol (PEG), such as a reactiveester or aldehyde derivative of PEG, under conditions in which one ormore PEG groups become attached to the antibody or antibody fragment.Typically, the pegylation is carried out via an acylation reaction or analkylation reaction with a reactive PEG molecule (or an analogousreactive water-soluble polymer). As used herein, the term “polyethyleneglycol” is intended to encompass any of the forms of PEG that have beenused to derivatize other proteins, such as mono (C₁ to C₁₀) alkoxy- oraryloxy-polyethylene glycol or polyethylene glycol-maleimide. In certaincases, the antibody to be pegylated is an aglycosylated antibody.Methods for pegylating proteins are known in the art and can be appliedto the antibodies of the present disclosure. See for example, EuropeanPatent Nos. EP 0154316B1 and EP 0401384B1.

Production of Monoclonal Antibodies of the Disclosure

Monoclonal antibodies (mAbs) of the present disclosure can be producedby a variety of techniques, including conventional monoclonal antibodymethodology e.g., the standard somatic cell hybridization technique ofKohler and Milstein, Nature 256:495 (1975). Other techniques forproducing monoclonal antibodies also can be employed, e.g., viral oroncogenic transformation of B lymphocytes.

The preferred animal system for preparing hybridomas is the murinesystem. Hybridoma production in the mouse is a very well-establishedprocedure. Immunization protocols and techniques for isolation ofimmunized splenocytes for fusion are known in the art. Fusion partners(e.g., murine myeloma cells) and fusion procedures are also known.

Humanized antibodies of the present disclosure can be prepared based onthe sequence of a murine monoclonal antibody prepared as describedabove. DNA encoding the heavy and light chain immunoglobulins can beobtained from the murine hybridoma of interest and engineered to containnon-murine (e.g., human) immunoglobulin sequences using suitablemolecular biology techniques. For example, to create a chimericantibody, the murine variable regions can be linked to human constantregions using methods known in the art (see e.g., U.S. Pat. No.4,816,567). To create a humanized antibody, the murine CDR regions canbe inserted into a human framework using methods known in the art (seee.g., U.S. Pat. No. 5,225,539, and U.S. Pat. Nos. 5,530,101; 5,585,089;5,693,762 and 6,180,370).

In some cases, the antibodies of the disclosure are human monoclonalantibodies. Such human monoclonal antibodies directed against OPN can begenerated using transgenic or transchromosomic mice carrying parts ofthe human immune system rather than the mouse system. See, e.g. Tayloret al. (1992) Nucleic Acids Research 20:6287-6295 (1992); Chen et al.International Immunology 5: 647-656 (1993); Tuaillon et al., Proc. Natl.Acad. Sci. USA 90:3720-3724 (1993); Choi et al. Nature Genetics4:117-123 (1993); Chen, et al., EMBO J. 12:821-830 (1993); Tuaillon etal., J. Immunol. 152:2912-2920 (1994); Taylor et al., InternationalImmunology 6:579-591 (1994); and Fishwild et al., Nature Biotechnology14:845-851 (1996). See further, U.S. Pat. Nos. 5,545,806; 5,569,825;5,625,126; 5,633,425; 5,789,650; 5,877,397; 5,661,016;

5,814,318; 5,874,299; and 5,770,429; U.S. Pat. No. 5,545,807; and PCTPublication Nos. WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO98/24884, WO 01/14424 and WO 99/45962.

In another case, human antibodies of the disclosure can be raised usinga mouse that carries human immunoglobulin sequences on transgenes andtranschomosomes, such as a mouse that carries a human heavy chaintransgene and a human light chain transchromosome. Such mice aredescribed in detail in PCT Publication WO 02/43478.

Still further, alternative transgenic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseOPN antibodies of the disclosure. For example, an alternative transgenicsystem referred to as the Xenomouse (Abgenix, Inc.) can be used; suchmice are described in, for example, U.S. Pat. Nos. 5,939,598; 6,075,181;6,114,598; 6,150,584 and 6,162,963.

Moreover, alternative transchromosomic animal systems expressing humanimmunoglobulin genes are available in the art and can be used to raiseOPN antibodies of the disclosure. For example, mice carrying both ahuman heavy chain transchromosome and a human light chaintranschromosome, referred to as “TC mice” can be used; such mice aredescribed in Tomizuka et al., Proc. Natl. Acad. Sci. USA 97:722-727(2000). Furthermore, cows carrying human heavy and light chaintranschromosomes have been described in the art (Kuroiwa et al., NatureBiotechnology 20:889-894 (2002)) and can be used to raise OPN antibodiesof the disclosure.

Human monoclonal antibodies of the disclosure can also be prepared usingphage display methods for screening libraries of human immunoglobulingenes. Such phage display methods (e.g. HuCAL® Libraries as describedfurther in Example 1 and herein) for isolating human antibodies areestablished in the art. See for example: U.S. Pat. Nos. 5,223,409;5,403,484; 5,571,698; 5,427,908; 5,580,717; 5,969,108; 6,172,197;5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915 and 6,593,081.

Human monoclonal antibodies of the disclosure can also be prepared usingSCID mice into which human immune cells have been reconstituted suchthat a human antibody response can be generated upon immunization. Suchmice are described in, for example, U.S. Pat. Nos. 5,476,996 and5,698,767.

Nucleic Acid Molecules of the Disclosure

The present disclosure also relates to nucleic acid molecules thatencode antibodies disclosed herein. These sequences include, but are notlimited to, those nucleic acid molecules set forth in FIGS. 1A, 1C, 1E,1G, 1I, 1K, 2A, 2C, 2E, 2G, 2I, and 2K. The nucleic acids may be presentin whole cells, in a cell lysate, or in a partially purified orsubstantially pure form. A nucleic acid is “isolated” or “renderedsubstantially pure” when purified away from other cellular components orother contaminants, e.g., other cellular nucleic acids or proteins, byany suitable techniques, including alkaline/SDS treatment, CsCl banding,column chromatography, agarose gel electrophoresis and others. A nucleicacid of the disclosure can be, for example, DNA or RNA and may or maynot contain intronic sequences. Typically, the nucleic acid is a cDNAmolecule.

Nucleic acids of the disclosure can be obtained using any suitablemolecular biology techniques. For antibodies expressed by hybridomas,cDNAs encoding the light and heavy chains of the antibody made by thehybridoma can be obtained by PCR amplification or cDNA cloningtechniques. For antibodies obtained from an immunoglobulin gene library(e.g., using phage display techniques), nucleic acid encoding theantibody can be recovered from the library.

The isolated DNA encoding the V_(H) region can be converted to afull-length heavy chain gene by operatively linking the V_(H)-encodingDNA to another DNA molecule encoding heavy chain constant regions (CH1,CH2 and CH3). The sequences of human heavy chain constant region genesare known in the art (see e.g., Kabat et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242) and DNAfragments encompassing these regions can be obtained by standard PCRamplification. The heavy chain constant region can be an IgG1, IgG2,IgG3, IgG4, IgA, IgE, IgM or IgD constant region, but most preferably isan IgG1 or IgG4 constant region. The IgG1 constant region sequence canbe any of the various alleles or allotypes known to occur amongdifferent individuals, such as Gm(1), Gm(2), Gm(3), and Gm(17). Theseallotypes represent naturally occurring amino acid substitution in theIgG1 constant regions. For a Fab fragment heavy chain gene, theV_(H)-encoding DNA can be operatively linked to another DNA moleculeencoding only the heavy chain CH1 constant region.

The isolated DNA encoding the V_(L) region can be converted to afull-length light chain gene (as well as a Fab light chain gene) byoperatively linking the V_(L)-encoding DNA to another DNA moleculeencoding the light chain constant region, CL. The sequences of humanlight chain constant region genes are known in the art (see e.g., Kabatet al. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242) and DNA fragments encompassing these regions can beobtained by standard PCR amplification. The light chain constant regioncan be a kappa or lambda constant region.

To create a scFv gene, the V_(H)- and V_(L)-encoding DNA fragments areoperatively linked to another fragment encoding a flexible linker, e.g.,encoding the amino acid sequence (Gly₄ -Ser)₃, such that the V_(H) andV_(L) sequences can be expressed as a contiguous single-chain protein,with the V_(L) and V_(H) regions joined by the flexible linker (seee.g., Bird et al., Science 242:423-426 (1988); Huston et al., Proc.Natl. Acad. Sci. USA 85:5879-5883 (1988); and McCafferty et al., Nature348:552-554 (1990)).

Nucleic Acid Variants

Nucleic acid molecules of the disclosure are not limited to thesequences disclosed herein, but also include variants thereof. Nucleicacid variants within the disclosure may be described by reference totheir physical properties in hybridization. For example, the skilledworker will recognize that DNA can be used to identify its complementand, since DNA is double stranded, its equivalent or homolog, usingnucleic acid hybridization techniques. It also will be recognized thathybridization can occur with less than 100% complementarity. However,given appropriate choice of conditions, hybridization techniques can beused to differentiate among DNA sequences based on their structuralrelatedness to a particular probe. For guidance regarding suchconditions see, Sambrook and Russell, Molecular Cloning, A LaboratoryApproach, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (2001) andAusubel et al., Current Protocols in Molecular Biology, John Wiley &Sons, NY (2002).

Structural similarity between two polynucleotide sequences can beexpressed as a function of “stringency” of the conditions under whichthe two sequences will hybridize with one another. As used herein, theterm “stringency” refers to the extent that the conditions disfavorhybridization. Stringent conditions strongly disfavor hybridization, andonly the most structurally related molecules will hybridize to oneanother under such conditions. Conversely, non-stringent conditionsfavor hybridization of molecules displaying a lesser degree ofstructural relatedness. Hybridization stringency, therefore, directlycorrelates with the structural relationships of two nucleic acidsequences. The following relationships are useful in correlatinghybridization and relatedness (where T_(m) is the melting temperature ofa nucleic acid duplex):

-   -   a. T_(m)=69.3+0.41(G+C)%    -   b. The T_(m) of a duplex DNA decreases by 1° C. with every        increase of 1% in the number of mismatched base pairs.    -   c. (T_(m))_(μ2)−(T_(m))_(μ1)=18.5 log₁₀μ2/μ1        -   where μ1 and μ2 are the ionic strengths of two solutions.

Hybridization stringency is a function of many factors, includingoverall DNA concentration, ionic strength, temperature, probe size andthe presence of agents which disrupt hydrogen bonding. Factors promotinghybridization include high DNA concentrations, high ionic strengths, lowtemperatures, longer probe size and the absence of agents that disrupthydrogen bonding. Hybridization typically is performed in two phases:the “binding” phase and the “washing” phase.

First, in the binding phase, the probe is bound to the target underconditions favoring hybridization. Stringency is usually controlled atthis stage by altering the temperature. For high stringency, thetemperature is usually between 65° C. and 70° C., unless short (<20nucleotides) oligonucleotide probes are used. A representativehybridization solution comprises 6×SSC, 0.5% SDS, 5× Denhardt's solutionand 100 μg of nonspecific carrier DNA. See Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (2002). Of course,many different, yet functionally equivalent, buffer conditions areknown. Where the degree of relatedness is lower, a lower temperature maybe chosen. Low stringency binding temperatures are between about 25° C.and 40° C. Medium stringency is between at least about 40° C. to lessthan about 65° C. High stringency is at least about 65° C.

Second, the excess probe is removed by washing. It is at this phase thatmore stringent conditions usually are applied. Hence, it is this“washing” stage that is most important in determining relatedness viahybridization. Washing solutions typically contain lower saltconcentrations. One exemplary medium stringency solution contains 2×SSCand 0.1% SDS. A high stringency wash solution contains the equivalent(in ionic strength) of less than about 0.2×SSC, with a preferredstringent solution containing about 0.1×SSC. The temperatures associatedwith various stringencies are the same as discussed above for “binding.”The washing solution also typically is replaced a number of times duringwashing. For example, typical high stringency washing conditionscomprise washing twice for 30 minutes at 55° C., and three times for 15minutes at 60° C.

Accordingly, the present disclosure includes nucleic acid molecules thathybridize to the DNA molecules as described herein under high stringencybinding and washing conditions, where such nucleic molecules encode anantibody or functional fragment thereof having properties as describedherein. Preferred molecules (from an mRNA perspective) are those thathave at least 75% or 80% (preferably at least 85%, more preferably atleast 90% and most preferably at least 95%) sequence identity with oneof the DNA molecules described herein.

Yet another class of nucleic acid variants within the scope of thepresent disclosure may be described with reference to the product theyencode. These functionally equivalent genes are characterized by thefact that they encode the same peptide sequences disclosed herein (e.g.in FIGS. 1 and 2) due to the degeneracy of the genetic code.

It is recognized that variants of DNA molecules provided herein can beconstructed in several different ways. For example, they may beconstructed as completely synthetic DNAs. Methods of efficientlysynthesizing oligonucleotides in the range of 20 to about 150nucleotides are widely available. See, e.g. Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (2002).Overlapping oligonucleotides may be synthesized and assembled in afashion first reported by Khorana et al., J. Mol. Biol. 72:209-217(1971). Synthetic DNAs preferably are designed with convenientrestriction sites engineered at the 5′ and 3′ ends of the gene tofacilitate cloning into an appropriate vector.

As indicated, a method of generating variants is to start with one ofthe DNAs disclosed herein and then to conduct site-directed mutagenesis.See Ausubel et al., Current Protocols in Molecular Biology, John Wiley &Sons, NY (2002). In a typical method, a target DNA is cloned into asingle-stranded DNA bacteriophage vehicle. Single-stranded DNA isisolated and hybridized with an oligonucleotide containing the desirednucleotide alteration(s). The complementary strand is synthesized andthe double stranded phage is introduced into a host. Some of theresulting progeny will contain the desired mutant, which can beconfirmed using DNA sequencing. In addition, various methods areavailable that increase the probability that the progeny phage will bethe desired mutant. These methods are well known to those in the fieldand kits are commercially available for generating such mutants.

Recombinant Nucleic Acid Constructs and Expression

The present disclosure further provides recombinant DNA constructscomprising one or more of the nucleotide sequences of the presentdisclosure. The recombinant constructs of the present disclosure areused in connection with a vector, such as a plasmid, phagemid, phage orviral vector, into which a DNA molecule encoding an antibody of thedisclosure is inserted.

The encoded gene may be produced by techniques described in Sambrook andRussell, Molecular Cloning, A Laboratory Approach, Cold Spring HarborPress, Cold Spring Harbor, N.Y. (2001), and Ausubel et al., CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (2002).Alternatively, the DNA sequences may be chemically synthesized using,for example, synthesizers. See, for example, the techniques described inOligonucleotide Synthesis (1984, Gait, ed., IRL Press, Oxford). Forexample, to express the antibodies of the disclosure, or antibodyfragments thereof, DNAs encoding partial or full-length light and heavychains, can be obtained by standard molecular biology techniques (e.g.,PCR amplification or cDNA cloning using a hybridoma or phage thatexpresses the antibody of interest) and the DNAs can be inserted intoexpression vectors such that the genes are operatively linked totranscriptional and translational control sequences. In this context,the term “operatively linked” is intended to mean that an antibody geneis ligated into a vector such that transcriptional and translationalcontrol sequences within the vector serve their intended function ofregulating the transcription and translation of the antibody gene. Theexpression vector and expression control sequences are chosen to becompatible with the expression host cell used. The antibody light chaingene and the antibody heavy chain gene can be inserted into separatevector or, more typically, both genes are inserted into the sameexpression vector. The antibody genes are inserted into the expressionvector by any suitable methods (e.g., ligation of complementaryrestriction sites on the antibody gene fragment and vector, or blunt endligation if no restriction sites are present). The light and heavy chainvariable regions of the antibodies described herein can be used tocreate full-length antibody genes of any antibody isotype and subclassby inserting them into expression vectors already encoding heavy chainconstant and light chain constant regions of the desired isotype andsubclass such that the V_(H) segment is operatively linked to the C_(H)segment(s) within the vector and the V_(K) segment is operatively linkedto the C_(L) segment within the vector. Additionally or alternatively,the recombinant expression vector can encode a signal peptide thatfacilitates secretion of the antibody chain from a host cell. Theantibody chain gene can be cloned into the vector such that the signalpeptide is linked in-frame to the amino terminus of the antibody chaingene. The signal peptide can be an immunoglobulin signal peptide or aheterologous signal peptide (i.e., a signal peptide from anon-immunoglobulin protein).

In addition to the antibody chain genes, the recombinant expressionvectors of the disclosure typically carry regulatory sequences thatcontrol the expression of the antibody chain genes in a host cell. Theterm “regulatory sequence” is intended to include promoters, enhancersand other expression control elements (e.g., polyadenylation signals)that control the transcription or translation of the antibody chaingenes. Such regulatory sequences are described, for example, in Goeddel(Gene Expression Technology. Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990)). It will be appreciated by those skilled inthe art that the design of the expression vector, including theselection of regulatory sequences, may depend on such factors as thechoice of the host cell to be transformed, the level of expression ofprotein desired, etc. Preferred regulatory sequences for mammalian hostcell expression include viral elements that direct high levels ofprotein expression in mammalian cells, such as promoters and/orenhancers derived from cytomegalovirus (CMV), Simian Virus 40 (SV40),adenovirus, (e.g., the adenovirus major late promoter (AdMLP) andpolyoma. Alternatively, nonviral regulatory sequences may be used, suchas the ubiquitin promoter or β-globin promoter. Still further,regulatory elements composed of sequences from different sources, suchas the SR promoter system, which contains sequences from the SV40 earlypromoter and the long terminal repeat of human T cell leukemia virustype 1 (Takebe, Y. et al. (1988) Mol. Cell. Biol. 8:466-472).

In addition to the antibody chain genes and regulatory sequences, therecombinant expression vectors of the disclosure may carry additionalsequences, such as sequences that regulate replication of the vector inhost cells (e.g., origins of replication) and selectable marker genes.The selectable marker gene facilitates selection of host cells intowhich the vector has been introduced (see, e.g., U.S. Pat. Nos.4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). For example,typically the selectable marker gene confers resistance to drugs, suchas G418, hygromycin or methotrexate, on a host cell into which thevector has been introduced. Selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in dhfr- host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

For expression of the light and heavy chains, the expression vector(s)encoding the heavy and light chains is transfected into a host cell byany suitable techniques. The various forms of the term “transfection”are intended to encompass a wide variety of techniques commonly used forthe introduction of exogenous DNA into a prokaryotic or eukaryotic hostcell, e.g., electroporation, calcium-phosphate precipitation,DEAE-dextran transfection and the like. Although it is possible toexpress the antibodies of the disclosure in either prokaryotic oreukaryotic host cells, expression of antibodies in eukaryotic cells, andtypically mammalian host cells, is most typical.

The present disclosure further provides host cells containing at leastone of the DNAs disclosed herein. The host cell can be virtually anycell for which expression vectors are available. It may be, for example,a higher eukaryotic host cell, such as a mammalian cell, a lowereukaryotic host cell, such as a yeast cell, and may be a prokaryoticcell, such as a bacterial cell. Introduction of the recombinantconstruct into the host cell can be effected by calcium phosphatetransfection, DEAE, dextran mediated transfection, electroporation orphage infection.

Useful expression vectors for bacterial use are constructed by insertinga structural DNA sequence encoding a desired protein together withsuitable translation initiation and termination signals in operablereading phase with a functional promoter. The vector will comprise oneor more phenotypic selectable markers and an origin of replication toensure maintenance of the vector and, if desirable, to provideamplification within the host. Suitable prokaryotic hosts fortransformation include E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus.

Bacterial vectors may be, for example, bacteriophage-, plasmid- orphagemid-based. These vectors can contain a selectable marker andbacterial origin of replication derived from commercially availableplasmids typically containing elements of the well known cloning vectorpBR322 (ATCC 37017). Following transformation of a suitable host strainand growth of the host strain to an appropriate cell density, theselected promoter is de-repressed/induced by appropriate means (e.g.,temperature shift or chemical induction) and cells are cultured for anadditional period. Cells are typically harvested by centrifugation,disrupted by physical or chemical means, and the resulting crude extractretained for further purification.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the proteinbeing expressed. For example, when a large quantity of such a protein isto be produced, for the generation of antibodies or to screen peptidelibraries, for example, vectors which direct the expression of highlevels of fusion protein products that are readily purified may bedesirable.

Mammalian host cells for expressing the recombinant antibodies of thedisclosure include, for example, Chinese Hamster Ovary (CHO) cells(including dhfr- CHO cells, described in Urlaub and Chasin, Proc. Natl.Acad. Sci. USA 77:4216-4220 (1980), used with a DHFR selectable marker,e.g., as described in Kaufman and Sharp, J. Mol. Biol. 159:601-621(1982), NS0 myeloma cells, COS cells and Sp2 cells. In particular, foruse with NS0 myeloma or CHO cells, another expression system is the GS(glutamine synthetase) gene expression system disclosed in WO 87/04462,WO 89/01036 and EP 338,841. When recombinant expression vectors encodingantibody genes are introduced into mammalian host cells, the antibodiesare produced by culturing the host cells for a period of time sufficientto allow for expression of the antibody in the host cells or secretionof the antibody into the culture medium in which the host cells aregrown. Antibodies can be recovered from the culture medium using anysuitable protein purification methods.

Immunoconjugates

In another aspect, the present disclosure features an OPN antibody, or afragment thereof, conjugated to a therapeutic moiety, such as acytotoxin, a drug (e.g., an immunosuppressant) or a radiotoxin. Suchconjugates are referred to herein as “immunoconjugates”.Immunoconjugates that include one or more cytotoxins are referred to as“immunotoxins.” A cytotoxin or cytotoxic agent includes any agent thatis detrimental to (e.g., kills) cells. Examples include taxol,cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin,etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin,daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin,actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine,tetracaine, lidocaine, propranolol, and puromycin and analogs orhomologs thereof. Therapeutic agents also include, for example,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

Other examples of therapeutic cytotoxins that can be conjugated to anantibody, or antigen binding portion thereof, of the disclosure includeduocarmycins, calicheamicins, maytansines and auristatins, andderivatives thereof. An example of a calicheamicin antibody conjugate iscommercially available (Mylotarg™; Wyeth-Ayerst).

Cytoxins can be conjugated to antibodies of the disclosure or antigenbinding portions thereof using various linker technologies. Examples oflinker types that have been used to conjugate a cytotoxin to an antibodyinclude, but are not limited to, hydrazones, thioethers, esters,disulfides and peptide-containing linkers. A linker can be chosen thatis, for example, susceptible to cleavage by low pH within the lysosomalcompartment or susceptible to cleavage by proteases, such as proteasespreferentially expressed in tumor tissue such as cathepsins (e.g.,cathepsins B, C, D).

For further discussion of types of cytotoxins, linkers and methods forconjugating therapeutic agents to antibodies, see also Saito et al.,Adv. Drug Deliv. Rev. 55:199-215 (2003); Trail, et al., Cancer Immunol.Immunother. 52:328-337 (2003); Payne, Cancer Cell 3:207-212 (2003);Allen, Nat. Rev. Cancer 2:750-763 (2002); Pastan, I. and Kreitman, Curr.Opin. Investig. Drugs 3:1089-1091 (2002); Senter, P. D. and Springer, C.J. (2001) Adv. Drug Deliv. Rev. 53:247-264.

Antibodies or antigen binding portions thereof of the present disclosurealso can be conjugated to a radioactive isotope to generate cytotoxicradiopharmaceuticals, also referred to as radioimmunoconjugates.Examples of radioactive isotopes that can be conjugated to antibodiesfor use diagnostically or therapeutically include, but are not limitedto, iodine¹³¹, indium¹¹¹, yttrium⁹⁰ and lutetium¹⁷⁷. Methods forpreparing radioimmunoconjugates are established in the art. Examples ofradioimmunoconjugates are commercially available, including Zevalin™(IDEC Pharmaceuticals) and Bexxar™ (Corixa Pharmaceuticals), and similarmethods can be used to prepare radioimmunoconjugates using theantibodies of the disclosure.

The antibody conjugates of the disclosure can be used to modify a givenbiological response, and the drug moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, the drugmoiety may be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, an enzymaticallyactive toxin, or active fragment thereof, such as abrin, ricin A,pseudomonas exotoxin, or diphtheria toxin; a protein such as tumornecrosis factor or interferon-γ; or, biological response modifiers suchas, for example, lymphokines, interleukin-1 (“IL-1”), interleukin-2(“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colonystimulating factor (“GM-CSF”), granulocyte colony stimulating factor(“G-CSF”), or other growth factors.

Techniques for conjugating such therapeutic moiety to antibodies areknown. See, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-256 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-653(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-316 (Academic Press 1985), and Thorpe et al., Immunol.Rev., 62:119-158 (1982).

Bispecific Molecules

In another aspect, the present disclosure features bispecific moleculescomprising an OPN antibody, or an antigen-binding portion thereof, ofthe present disclosure. An antibody of the disclosure, orantigen-binding portion thereof, can be derivatized or linked to anotherfunctional molecule, e.g., another peptide or protein (e.g., anotherantibody or ligand for a receptor) to generate a bispecific moleculethat binds to at least two different binding sites or target molecules.The antibodies of the disclosure may in fact be derivatized or linked tomore than one other functional molecule to generate multispecificmolecules that bind to more than two different binding sites and/ortarget molecules; such multispecific molecules are also intended to beencompassed by the term “bispecific molecule” as used herein. To createa bispecific molecule of the disclosure, an antibody of the disclosurecan be functionally linked (e.g., by chemical coupling, genetic fusion,noncovalent association or otherwise) to one or more other bindingmolecules, such as another antibody, antibody fragment, peptide orbinding mimetic, such that a bispecific molecule results.

Accordingly, the present disclosure includes bispecific moleculescomprising at least one first binding specificity for OPN and a secondbinding specificity for a second target epitope. In a particular aspectof the disclosure, the second target epitope is an Fc receptor, e.g.,human FcγRI (CD64) or a human Fcγ receptor (CD89). Therefore, thedisclosure includes bispecific molecules capable of binding both to FcγRor FcγR expressing effector cells (e.g., monocytes, macrophages orpolymorphonuclear cells (PMNs)), and to OPN. These bispecific moleculestarget OPN to effector cell and trigger Fc receptor-mediated effectorcell activities, such as phagocytosis of an OPN expressing cell,antibody dependent cell-mediated cytotoxicity (ADCC), cytokine release,or generation of superoxide anion.

In an aspect of the disclosure in which the bispecific molecule ismultispecific, the molecule can further include a third bindingspecificity, in addition to an anti-Fc binding specificity and an OPNbinding specificity. In one case, the third binding specificity is ananti-enhancement factor (EF) portion, e.g., a molecule which binds to asurface protein involved in cytotoxic activity and thereby increases theimmune response against the target cell. The “anti-enhancement factorportion” can be an antibody, functional antibody fragment or a ligandthat binds to a given molecule, e.g., an antigen or a receptor, andthereby results in an enhancement of the effect of the bindingdeterminants for the Fc receptor or target cell antigen. The“anti-enhancement factor portion” can bind an Fc receptor or a targetcell antigen. Alternatively, the anti-enhancement factor portion canbind to an entity that is different from the entity to which the firstand second binding specificities bind. For example, the anti-enhancementfactor portion can bind a cytotoxic T-cell (e.g. via CD2, CD3, CD8,CD28, CD4, CD40, ICAM-1 or other immune cell that results in anincreased immune response against the target cell).

In one case, the bispecific molecules of the disclosure comprise as abinding specificity at least one antibody, or an antibody fragmentthereof, including, e.g., an Fab, Fab′, F(ab′)₂, Fv, or a single chainFv. The antibody may also be a light chain or heavy chain dimer, or anyminimal fragment thereof such as a Fv or a single chain construct asdescribed in U.S. Pat. No. 4,946,778.

In one case, the binding specificity for an Fcγ receptor is provided bya monoclonal antibody, the binding of which is not blocked by humanimmunoglobulin G (IgG). As used herein, the term “IgG receptor” refersto any of the eight γ-chain genes located on chromosome 1. These genesencode a total of twelve transmembrane or soluble receptor isoformswhich are grouped into three Fcγ receptor classes: FcγRI (CD64),FcγRII(CD32), and FcγRIII (CD16). In one case, the Fcγ receptor is ahuman high affinity FcγRI. The human FcγRI is a 72 kDa molecule, whichshows high affinity for monomeric IgG (10⁸ to 10⁹ M⁻¹).

The production and characterization of certain anti-Fcy monoclonalantibodies are described in PCT Publication WO 88/00052 and in U.S. Pat.No. 4,954,617. These antibodies bind to an epitope of FcγRI, FcγRII orFcγRIII at a site which is distinct from the Fcγ binding site of thereceptor and, thus, their binding is not blocked substantially byphysiological levels of IgG. Specific anti-FcγRI antibodies useful inthis disclosure are mAb 22, mAb 32, mAb 44, mAb 62 and mAb 197. Thehybridoma producing mAb 32 is available from the American Type CultureCollection, ATCC Accession No. HB9469. In other cases the anti-Fcγreceptor antibody is a humanized form of monoclonal antibody 22 (H22).The production and characterization of the H22 antibody is described inGraziano et al. J. Immunol 155(10):4996-5002 (1995) and PCT PublicationWO 94/10332. The H22 antibody producing cell line was deposited at theAmerican Type Culture Collection under the designation HA022CL1 and hasthe Accession No. CRL 11177.

In still other cases, the binding specificity for an Fc receptor isprovided by an antibody that binds to a human IgA receptor, e.g., anFc-alpha receptor (FcαRI (CD89)), the binding of which is typically notblocked by human immunoglobulin A (IgA). The term “IgA receptor” isintended to include the gene product of one α-gene (FcαRI) located onchromosome 19. This gene is known to encode several alternativelyspliced transmembrane isoforms of 55 to 110 kDa. FcαRI (CD89) isconstitutively expressed on monocytes/macrophages, eosinophilic andneutrophilic granulocytes, but not on non-effector cell populations.FcαRI has medium affinity 5×10⁷ M⁻¹) for both IgA1 and IgA2, which isincreased upon exposure to cytokines such as G-CSF or GM-CSF (Morton etal., Critical Reviews in Immunology 16:423-440 (1996)). FourFcαRI-specific monoclonal antibodies, identified as A3, A59, A62 andA77, which bind FcαRI outside the IgA ligand binding domain, have beendescribed (Monteiro et al., J. Immunol. 148:1764 (1992)).

FcαRI and FcγRI are illustrative trigger receptors for use in thebispecific molecules of the disclosure because they are (1) expressedprimarily on immune effector cells, e.g., monocytes, PMNs, macrophagesand dendritic cells; (2) expressed at high levels (e.g., 5,000 to100,000 per cell); (3) mediators of cytotoxic activities (e.g., ADCC,phagocytosis); (4) mediate enhanced antigen presentation of antigens,including self-antigens, targeted to them.

While human monoclonal antibodies are preferred, other antibodies whichcan be employed in the bispecific molecules of the disclosure aremurine, chimeric and humanized monoclonal antibodies.

The bispecific molecules of the present disclosure can be prepared byconjugating the constituent binding specificities, e.g., the anti-FcRand anti-OPN binding specificities, using any suitable methods. Forexample, each binding specificity of the bispecific molecule can begenerated separately and then conjugated to one another. When thebinding specificities are proteins or peptides, a variety of coupling orcross-linking agents can be used for covalent conjugation. Examples ofcross-linking agents include protein A, carbodiimide,N-succinimidyl-S-acetyl-thioacetate (SATA),5,5′-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide(oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), andsulfosuccinimidyl 4-(N-maleimidomethyl)cyclohaxane-1-carboxylate(sulfo-SMCC) (see e.g., Karpovsky et al., J. Exp. Med. 160:1686 (1984);Liu et al., Proc. Natl. Acad. Sci. USA 82:8648 (1985)). Other methodsinclude those described in Paulus (1985) Behring Ins. Mitt. No. 78,118-132; Brennan et al., Science 229:81-83 (1985)), and Glennie et al.,J. Immunol. 139:2367-2375 (1987)). Suitable conjugating agents includeSATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford,Ill.).

When the binding specificities are antibodies, they can be conjugatedvia sulfhydryl bonding of the C-terminus hinge regions of the two heavychains. In one case, the hinge region is modified to contain an oddnumber of sulfhydryl residues, such as one residue, prior toconjugation.

Alternatively, both binding specificities can be encoded in the samevector and expressed and assembled in the same host cell. This method isparticularly useful where the bispecific molecule is a mAb×mAb, mAb×Fab,Fab×F(ab′)₂ or ligand×Fab fusion protein. A bispecific molecule of thedisclosure can be a single chain molecule comprising one single chainantibody and a binding determinant, or a single chain bispecificmolecule comprising two binding determinants. Bispecific molecules maycomprise at least two single chain molecules. Methods for preparingbispecific molecules are described for example in U.S. Pat. Nos.5,260,203; 5,455,030; 4,881,175; 5,132,405; 5,091,513; 5,476,786;5,013,653; 5,258,498; and 5,482,858.

Binding of the bispecific molecules to their specific targets can beconfirmed by, for example, enzyme-linked immunosorbent assay (ELISA),radioimmunoassay (RIA), FACS analysis, bioassay (e.g., growthinhibition), or Western Blot assay. Each of these assays generallydetects the presence of protein-antibody complexes of particularinterest by employing a labeled reagent (e.g., an antibody) specific forthe complex of interest. For example, the FcR-antibody complexes can bedetected using e.g., an enzyme-linked antibody or antibody fragmentwhich recognizes and specifically binds to the antibody-FcR complexes.Alternatively, the complexes can be detected using any of a variety ofother immunoassays. For example, the antibody can be radioactivelylabeled and used in a radioimmunoassay (RIA) (see, for example,Weintraub, B., Principles of Radioimmunoassays, Seventh Training Courseon Radioligand Assay Techniques, The Endocrine Society, March, 1986).The radioactive isotope can be detected by such means as the use of a γcounter or a scintillation counter or by autoradiography.

Therapeutic Methods

Therapeutic methods involve administering to a subject in need oftreatment a therapeutically effective amount, or “effective amount”, ofan antibody, or antigen-binding portion, contemplated by the presentdisclosure. As used herein, a “therapeutically effective”, or“effective”, amount refers to an amount of an antibody or portionthereof that is of sufficient quantity to result in a decrease inseverity of disease symptoms, an increase in frequency and duration ofdisease symptom-free periods, or a prevention of impairment ordisability due to the disease affliction—either as a single dose oraccording to a multiple dose regimen, alone or in combination with otheragents. One of ordinary skill in the art would be able to determine suchamounts based on such factors as the subject's size, the severity of thesubject's symptoms, and the particular composition or route ofadministration selected. The subject may be a human or non-human animal(e.g., rabbit, rat, mouse, monkey or other lower-order primate).

An antibody or antigen-binding portion of the disclosure might beco-administered with known medicaments, and in some instances theantibody might itself be modified. For example, an antibody could beconjugated to an immunotoxin or radioisotope to potentially furtherincrease efficacy. Regarding co-administration with additionaltherapeutic agents, such agents can include a cytotoxic agent, aradiotoxic agent or an immunosuppressive agent. The antibody can belinked to the agent (as an immunocomplex) or can be administeredseparately from the agent. In the latter case (separate administration),the antibody can be administered before, after or concurrently with theagent or can be co-administered with other known therapies, e.g., ananti-cancer therapy, e.g., radiation. Such therapeutic agents include,among others, anti-neoplastic agents such as doxorubicin (adriamycin),cisplatin bleomycin sulfate, carmustine, chlorambucil, andcyclophosphamide hydroxyurea which, by themselves, are only effective atlevels which are toxic or subtoxic to a patient. Cisplatin can beintravenously administered as a 100 mg dose once every four weeks andadriamycin is intravenously administered as a 60 to 75 mg dose onceevery 21 days. Co-administration of the OPN antibodies, or antigenbinding fragments thereof, of the present disclosure withchemotherapeutic agents provides two anti-cancer agents which operatevia different mechanisms which yield a cytotoxic effect to human tumorcells. Such co-administration can solve problems due to development ofresistance to drugs or a change in the antigenicity of the tumor cellswhich would render them unreactive with the antibody.

The antibodies and antigen-binding portions disclosed herein can be usedas a therapeutic or a diagnostic tool in a variety of situations whereOPN is undesirably expressed or found. Given the expression of OPN byvarious tumor cells, and the role that OPN plays in tumor metastasis,disorders and conditions particularly suitable for treatment with anantibody or antigen-binding portion of the present disclosure includeabnormal cell growth, for example, mesothelioma, hepatobilliary (hepaticand billiary duct), a primary or secondary CNS tumor, a primary orsecondary brain tumor, lung cancer (NSCLC and SCLC), bone cancer,pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous orintraocular melanoma, ovarian cancer, colon cancer, rectal cancer,cancer of the anal region, stomach cancer, gastrointestinal (gastric,colorectal, and duodenal), breast cancer, uterine cancer, carcinoma ofthe fallopian tubes, carcinoma of the endometrium, carcinoma of thecervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin'sDisease, cancer of the esophagus, cancer of the small intestine, cancerof the endocrine system, cancer of the thyroid gland, cancer of theparathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue,cancer of the urethra, cancer of the penis, prostate cancer, testicularcancer, chronic or acute leukemia, chronic myeloid leukemia, lymphocyticlymphomas, cancer of the bladder, cancer of the kidney or ureter, renalcell carcinoma, carcinoma of the renal pelvis, neoplasms of the centralnervous system (CNS), primary CNS lymphoma, non-Hodgkin's lymphoma,spinal axis tumors, brain stem glioma, pituitary adenoma, adrenocorticalcancer, gall bladder cancer, multiple myeloma, cholangiocarcinoma,fibrosarcoma, neuroblastoma, retinoblastoma, or a combination of one ormore of the foregoing cancers.

To treat any of the foregoing disorders, pharmaceutical compositions foruse in accordance with the present disclosure may be formulated in aconventional manner using one or more pharmaceutically acceptablecarriers or excipients. An antibody or antigen-binding portion of thedisclosure can be administered by any suitable means, which can vary,depending on the type of disorder being treated. Possible administrationroutes include parenteral (e.g., intramuscular, intravenous,intra-arterial, intraperitoneal, or subcutaneous), intrapulmonary andintranasal, and, if desired for local immunosuppressive treatment,intralesional administration. In addition, an antibody of the disclosuremight be administered by pulse infusion, with, e.g., declining doses ofthe antibody. Preferably, the dosing is given by injections, mostpreferably intravenous or subcutaneous injections, depending in part onwhether the administration is brief or chronic. The amount to beadministered will depend on a variety of factors such as the clinicalsymptoms, weight of the individual, whether other drugs areadministered. The skilled artisan will recognize that the route ofadministration will vary depending on the disorder or condition to betreated.

Determining a therapeutically effective amount of an antibody orantigen-binding portion according to the present disclosure will largelydepend on particular patient characteristics, route of administration,and the nature of the disorder being treated. General guidance can befound, for example, in the publications of the International Conferenceon Harmonization and in Remington's Pharmaceutical Sciences, chapters 27and 28, pp. 484-528 (18th ed., Alfonso R. Gennaro, Ed., Easton, Pa.:Mack Pub. Co., 1990). More specifically, determining a therapeuticallyeffective amount will depend on such factors as toxicity and efficacy ofthe medicament. Toxicity may be determined using methods well known inthe art and found in the foregoing references. Efficacy may bedetermined utilizing the same guidance in conjunction with the methodsdescribed below in the Examples.

For administration of the antibody, the dosage can range from about0.0001 to 100 mg/kg, and more usually 0.01 to 20 mg/kg, of the host bodyweight. For example dosages can be 0.3 mg/kg body weight, 1 mg/kg bodyweight, 3 mg/kg body weight, 5 mg/kg body weight, 10 mg/kg body weight,15 mg/kg body weight, 20 mg/kg body weight, or within the range of 1 to20 mg/kg. An exemplary treatment regime entails administration once perweek, once every two weeks, once every three weeks, once every fourweeks, once per month, once every 3 months or once every three to 6months. Dosage regimens for an anti-OPN antibody or antigen bindingportion thereof of the disclosure include, for example, 1 mg/kg bodyweight, 3 mg/kg body weight, 5 mg/kg body weight, 10 mg/kg body weight,15 mg/kg body weight, or 20 mg/kg body weight via intravenousadministration, with the antibody being given using one of the followingdosing schedules: (i) every four weeks for six dosages, then every threemonths; (ii) every three weeks; (iii) 1-20 mg/kg body weight oncefollowed by 1-20 mg/kg body weight every three weeks.

Diagnostic Methods

OPN is highly expressed in various tumor cells; thus, an anti-OPNantibody of the disclosure may be employed in order to image orvisualize a site of possible OPN in a patient. In this regard, anantibody can be detectably labeled, through the use of radioisotopes,affinity labels (such as biotin, avidin, etc.) fluorescent labels,paramagnetic atoms, etc. Procedures for accomplishing such labeling arewell known to the art. Clinical application of antibodies in diagnosticimaging are reviewed by Grossman, Urol. Clin. North Amer. 13:465-474(1986), Unger et al., Invest. Radiol. 20:693-700 (1985), and Khaw etal., Science 209:295-297 (1980).

The detection of foci of such detectably labeled antibodies might beindicative of certain types of cancer, for example. In one embodiment,this examination is done by removing samples of tissue or blood andincubating such samples in the presence of the detectably labeledantibodies. In a preferred embodiment, this technique is done in anon-invasive manner through the use of magnetic imaging, fluorography,etc. Such a diagnostic test may be employed in monitoring the success oftreatment of diseases, where presence or absence of a targetOPN-positive cell is a relevant indicator.

Therapeutic and Diagnostic Compositions

The antibodies and antigen-binding portions of the present disclosurecan be formulated according to known methods to prepare pharmaceuticallyuseful compositions, wherein at least one antibody of the presentdisclosure (including any antigen-binding portion thereof) is combinedin a mixture with a pharmaceutically acceptable carrier. Suitablecarriers and their formulation are described, for example, inRemington's Pharmaceutical Sciences (18th ed., Alfonso R. Gennaro, Ed.,Easton, Pa.: Mack Pub. Co., 1990). In order to form a pharmaceuticallyacceptable composition suitable for effective administration, suchcompositions will contain an effective amount of one or more of theantibodies of the present disclosure, together with a suitable amount ofa pharmaceutically acceptable carrier.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. Typically, the carrier is suitable forintravenous, intramuscular, subcutaneous, parenteral, spinal orepidermal administration (e.g., by injection or infusion). Depending onthe route of administration, the active compound, i.e., antibody,antigen-binding portion thereof, immunoconjugate, or bispecificmolecule, may be coated in a material to protect the compound from theaction of acids and other natural conditions that may inactivate thecompound.

In certain embodiments, the antibodies of the present disclosure may bepresent in a neutral form (including zwitter ionic forms) or as apositively or negatively-charged species. In some cases, the antibodiesmay be complexed with a counterion to form a pharmaceutically acceptablesalt. Thus, the pharmaceutical compounds of the disclosure may includeone or more pharmaceutically acceptable salts.

A “pharmaceutically acceptable salt” refers to a salt that retains thedesired biological activity of the parent compound (e.g. antibody) anddoes not impart undesired toxicological effects (see e.g., Berge, S. M.,et al. (1977) J. Pharm. Sci. 66:1-19). For example, the term“pharmaceutically acceptable salt” includes a complex comprising one ormore antibodies and one or more counterions, where the counterions arederived from pharmaceutically acceptable inorganic and organic acids andbases.

Examples of such salts include acid addition salts and base additionsalts. Acid addition salts include those derived from nontoxic inorganicacids, such as hydrochloric, nitric, phosphoric, sulfuric, hydrobromic,hydroiodic, phosphorous and the like, as well as from nontoxic organicacids such as aliphatic mono- and dicarboxylic acids, phenyl-substitutedalkanoic acids, hydroxy alkanoic acids, aromatic acids, aliphatic andaromatic sulfonic acids and the like. Base addition salts include thosederived from alkaline earth metals, such as sodium, potassium,magnesium, calcium and the like, as well as from nontoxic organicamines, such as N,N′-dibenzylethylenediamine, N-methylglucamine,chloroprocaine, choline, diethanolamine, ethylenediamine, procaine andthe like.

Furthermore, pharmaceutically acceptable inorganic bases includemetallic ions. Metallic ions include, but are not limited to,appropriate alkali metal salts, alkaline earth metal salts and otherphysiological acceptable metal ions. Salts derived from inorganic basesinclude aluminum, ammonium, calcium, cobalt, nickel, molybdenum,vanadium, manganese, chromium, selenium, tin, copper, ferric, ferrous,lithium, magnesium, manganic salts, manganous, potassium, rubidium,sodium, and zinc, and in their usual valences.

Pharmaceutically acceptable acid addition salts of the antibodies of thepresent disclosure can be prepared from the following acids, including,without limitation formic, acetic, acetamidobenzoic, adipic, ascorbic,boric, propionic, benzoic, camphoric, carbonic, cyclamic, dehydrocholic,malonic, edetic, ethylsulfuric, fendizoic, metaphosphoric, succinic,glycolic, gluconic, lactic, malic, tartaric, tannic, citric, nitric,ascorbic, glucuronic, maleic, folic, fumaric, propionic, pyruvic,aspartic, glutamic, benzoic, hydrochloric, hydrobromic, hydroiodic,lysine, isocitric, trifluoroacetic, pamoic, propionic, anthranilic,mesylic, orotic, oxalic, oxalacetic, oleic, stearic, salicylic,aminosalicylic, silicate, p-hydroxybenzoic, nicotinic, phenylacetic,mandelic, embonic, sulfonic, methanesulfonic, phosphoric, phosphonic,ethanesulfonic, ethanedisulfonic, ammonium, benzenesulfonic,pantothenic, naphthalenesulfonic, toluenesulfonic,2-hydroxyethanesulfonic, sulfanilic, sulfuric, nitric, nitrous, sulfuricacid monomethyl ester, cyclohexylaminosulfonic, β-hydroxybutyric,glycine, glycylglycine, glutamic, cacodylate, diaminohexanoic,camphorsulfonic, gluconic, thiocyanic, oxoglutaric, pyridoxal5-phosphate, chlorophenoxyacetic, undecanoic, N-acetyl-L-aspartic,galactaric and galacturonic acids.

Pharmaceutically acceptable organic bases include trimethylamine,diethylamine, N, N′-dibenzylethylenediamine, chloroprocaine, choline,dibenzylamine, diethanolamine, ethylenediamine,meglumine(N-methylglucamine), procaine, cyclic amines, quaternaryammonium cations, arginine, betaine, caffeine, clemizole,2-ethylaminoethanol, 2-diethylaminoethanol, 2-dimethylaminoethanol,ethanediamine, butylamine, ethanolamine, ethylenediamine,N-ethylmorpholine, N-ethylpiperidine, ethylglucamine, glucamine,glucosamine, histidine, hydrabamine, imidazole, isopropylamine,methylglucamine, morpholine, piperazine, pyridine, pyridoxine,neodymium, piperidine, polyamine resins, procaine, purines, theobromine,triethylamine, tripropylamine, triethanolamine, tromethamine,methylamine, taurine, cholate, 6-amino-2-methyl-2-heptanol,2-amino-2-methyl-1,3-propanediol, 2-amino-2-methyl-1-propanol, aliphaticmono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, aliphatic and aromatic sulfonic acids,strontium, tricine, hydrazine, phenylcyclohexylamine,2-(N-morpholino)ethanesulfonic acid,bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane,N-(2-acetamido)-2-aminoethanesulfonic acid,1,4-piperazinediethanesulfonic acid,3-morpholino-2-hydroxypropanesulfonic acid,1,3-bis[tris(hydroxymethyl)methylamino]propane,4-morpholinepropanesulfonic acid,4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid,2-[(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid,N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid,4-(N-morpholino)butanesulfonic acid,3-(N,N-bis[2-hydroxyethyl]amino)-2-hydroxypropanesulfonic acid,2-hydroxy-3-[tris(hydroxymethyl)methylamino]-1-propanesulfonic acid,4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid),piperazine-1,4-bis(2-hydroxypropanesulfonic acid)dihydrate,4-(2-hydroxyethyl)-1-piperazinepropanesulfonic acid,N,N-bis(2-hydroxyethyl)glycine,N-(2-hydroxyethyl)piperazine-N′-(4-butanesulfonic acid),N-[tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid,N-tris(Hydroxymethyl)methyl-4-aminobutanesulfonic acid,N-(1,1-dimethyl-2-hydroxyethyl)-3-amino-2-hydroxypropanesulfonic acid,2-(cyclohexylamino)ethanesulfonic acid,3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid,3-(cyclohexylamino)-1-propanesulfonic acid, N-(2-acetamido)iminodiaceticacid, 4-(cyclohexylamino)-1-butanesulfonic acid,N-[tris(hydroxymethyl)methyl]glycine,2-amino-2-(hydroxymethyl)-1,3-propanediol, and trometamol.

A pharmaceutical composition of the disclosure also may include apharmaceutically acceptable anti-oxidant. Examples of pharmaceuticallyacceptable antioxidants include: (1) water soluble antioxidants, such asascorbic acid, cysteine hydrochloride, sodium bisulfate, sodiummetabisulfite, sodium sulfite and the like; (2) oil-solubleantioxidants, such as ascorbyl palmitate, butylated hydroxyanisole(BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate,alpha-tocopherol, and the like; and (3) metal chelating agents, such ascitric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaricacid, phosphoric acid, and the like.

Examples of suitable aqueous and nonaqueous carriers that may beemployed in the pharmaceutical compositions of the disclosure includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents and dispersing agents. Prevention ofpresence of microorganisms may be ensured both by sterilizationprocedures, supra, and by the inclusion of various antibacterial andantifungal agents, for example, paraben, chlorobutanol, phenol sorbicacid, and the like. It may also be desirable to include isotonic agents,such as sugars, sodium chloride, and the like into the compositions. Inaddition, prolonged absorption of the injectable pharmaceutical form maybe brought about by the inclusion of agents which delay absorption suchas aluminum monostearate and gelatin.

Pharmaceutically acceptable carriers include sterile aqueous solutionsor dispersions and sterile powders for the extemporaneous preparation ofsterile injectable solutions or dispersion. The use of such media andagents for pharmaceutically active substances is known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the pharmaceutical compositions ofthe disclosure is contemplated. Supplementary active compounds can alsobe incorporated into the compositions.

Therapeutic compositions typically must be sterile and stable under theconditions of manufacture and storage. The composition can be formulatedas a solution, microemulsion, liposome, or other ordered structuresuitable to high drug concentration. The carrier can be a solvent ordispersion medium containing, for example, water, ethanol, polyol (forexample, glycerol, propylene glycol, and liquid polyethylene glycol, andthe like), and suitable mixtures thereof. The proper fluidity can bemaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, polyalcohols such asmannitol, sorbitol, or sodium chloride in the composition. Prolongedabsorption of the injectable compositions can be brought about byincluding in the composition an agent that delays absorption, forexample, monostearate salts and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed bysterilization microfiltration. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation include, but arenot limited to, vacuum drying and freeze-drying (lyophilization) thatyield a powder of the active ingredient plus any additional desiredingredient from a previously sterile-filtered solution thereof.

The amount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will vary depending upon thesubject being treated, and the particular mode of administration. Theamount of active ingredient which can be combined with a carriermaterial to produce a single dosage form will generally be that amountof the composition which produces a therapeutic effect. Generally, outof one hundred percent, this amount will range from about 0.01 percentto about ninety-nine percent of active ingredient, preferably from about0.1 percent to about 70 percent, most preferably from about 1 percent toabout 30 percent of active ingredient in combination with apharmaceutically acceptable carrier.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofactive compound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the disclosure are dictated by and directlydependent on (a) the unique characteristics of the active compound andthe particular therapeutic effect to be achieved, and (b) thelimitations inherent in the art of compounding such an active compoundfor the treatment of sensitivity in individuals.

Preparations may be suitably formulated to provide controlled-release ofthe active compound. Controlled-release preparations may be achievedthrough the use of polymers to complex or absorb an anti-OPN antibody.The controlled delivery may be exercised by selecting appropriatemacromolecules (for example polyesters, polyamino acids, polyvinyl,pyrrolidone, ethylenevinyl-acetate, methylcellulose,carboxymethylcellulose, or protamine, sulfate) and the concentration ofmacromolecules as well as the methods of incorporation in order tocontrol release. Another possible method to control the duration ofaction by controlled release preparations is to incorporate an anti-OPNantibody into particles of a polymeric material such as polyesters,polyamino acids, hydrogels, poly(lactic acid) or ethylene vinylacetatecopolymers. Alternatively, instead of incorporating these agents intopolymeric particles, it is possible to entrap these materials inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatine-microcapsules and poly(methylmethacylate) microcapsules,respectively, or in colloidal drug delivery systems, for example,liposomes, albumin microspheres, microemulsions, nanoparticles, andnanocapsules or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences (18th ed., Alfonso R. Gennaro, Ed.,Easton, Pa.: Mack Pub. Co., 1990).

Antibody preparations may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampules, orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compositions may, if desired, be presented in a pack or dispenserdevice, which may contain one or more unit dosage forms containing theactive ingredient. The pack may, for example, comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

The present disclosure is further illustrated by the following exampleswhich should not be construed as further limiting. The contents of allfigures and all references, patents and published patent applicationscited throughout this disclosure are expressly incorporated herein byreference in their entirety.

EXAMPLES

As used in the Examples below, the following abbreviations have thefollowing meanings, unless indicated otherwise, are readily availablefrom commercial suppliers: PBS: phosphate buffered saline, pH 7.4; IPTG:Isopropyl-β-D-thiogalactopyranoside; HSA: human serum albumin;

Example 1 Antibody Generation from HuCAL® Libraries

For the generation of therapeutic antibodies against OPN, selectionswith the MorphoSys HuCAL GOLD® phagemid library were carried out. Thephagemid library is based on the HuCAL® concept (Knappik et al., J. Mol.Biol. 296:57 (2000)) and employs the CysDisplay™ technology fordisplaying the Fab on the phage surface (Löhning, WO 01/05950). HuCALGOLD® antibody-phage of different frameworks were either combined toform one pool (VH1-6) or were divided into sub-pools (e.g. VH1/5,VH2/4/6, VH3) and subsequently these sub-pools were individuallysubjected to selection rounds on antigen as described below. Phage forthe 1st round of pannings were prepared by Hyperphage (M13KO7ΔpIII,obtained from Progen, Heidelberg, Germany).

Solid Phase Panning Against OPN

Solid phase panning was performed using recombinant human OPN (R&DSystems #1433-OP/CF, carrier free), recombinant mouse OPN (R&D Systems#441-OP/CF, carrier free), or SPP1 peptides comprising functionaldomains of human OPN or mouse OPN (27 aa). Different antigens were usedfor either 3 rounds with or without alternating between human and mouseOPN as well as strategies including alternating of human/mouse OPNantigen with SPP1 peptides. For pannings using OPN proteins, differentbuffer conditions were used. Antigen was coated either in PBS or PBSsupplemented with Ca²⁺ and Mg²⁺ (100 mg/L CaCl₂; 100 mg/L MgCl₂).

A final concentration of 50 μg/mL diluted in PBS or PBS (supplementedwith Ca²⁺ and Mg²⁺) of human/mouse OPN was used for coating of anappropriate number of wells on Maxisorp™ plates (F96 Maxisorp™, 442402,Nunc, Rochester, N.Y.) for the first and second round of pannings. Forthe third round, 25 μg/mL of human and 10 μg/mL of mouse OPN were usedfor coating. Respective plates were then incubated overnight at 4° C. Onthe next day the wells were washed twice with PBS and then blocked withMPBST (PBS, 0.05% Tween20 (Sigma, St. Louis, Mo., USA), 5% milk powder)for 2 hours at room temperature. 100 μL of phage from original HuCALGOLD® subpools (VH1-6, VH1/5 and VH3, prepared with hyperphage) wereused. Phages were pre-blocked in a PBS solution containing 2.5% milkpowder, 2.5% BSA and 0.05% Tween20. The pre-blocking of phage wasperformed in 2 mL reaction tubes for 2 hours at room temperature on arotator.

For the selection process, the antigen solution was removed from theMaxisorp™ plate and the wells were washed three times with PBS. Thepre-blocked phage were added to the corresponding wells and the platewas incubated for 2 hours at room temperature on a microplate shaker.The phage solution was then removed and the wells were washed severaltimes (stringency depending on the panning strategy and selection round)with PBST (PBS, 0.05% Tween20), followed by the same washing steps withPBS. The washing stringency was increased from round to round. PBS wasremoved after the last washing step before continuing with elution. Forelution of specifically bound phage, 20 mM DTT in 10 mM Tris/HCl, pH 8.0was added and the samples were incubated for 10 minutes at roomtemperature.

The eluates were used to infect log phase E. coli TG1 cultures. InfectedE. coli were harvested by centrifugation and plated onto LB agar platessupplemented with 34 μg/mL chloramphenicol and 1% glucose. The agarplates were incubated overnight at 30° C. On the following day thecolonies were scraped off and grown until reaching an OD600 nm of 0.5 toproceed to helper phage infection. Helper phage infection: TG1 cellswere infected with the helper phage VCSM13 (multiplicity of infection ofat least 20) at 37° C. The infected cells were harvested bycentrifugation and resuspended in 2×YT medium containing 34 μg/mLchloramphenicol, 50 μg/mL kanamycin and 0.25 mM IPTG for induction ofFab expression. The cells were grown overnight and the produced phagewere precipitated from the supernatant with polyethylene glycol(PEG)/NaCl and resuspended in PBS. Input and output titers weredetermined by spot titration.

Solution Panning Against OPN

Solution panning was performed using recombinant human OPN, recombinantmouse OPN, or SPP1 peptides comprising functional domains of hOPN ormOPN (27aa). Different antigens were used for either 3 rounds with orwithout alternating between human and mouse OPN as well as strategiesincluding alternating of h/m OPN antigen with SPP1 peptides. Forpannings using OPN proteins, different buffer conditions were used.Antigen was used either in PBS or PBS supplemented with Ca²⁺ and Mg²⁺(100 mg/L CaCl₂; 100 mg/L MgCl₂).

All tubes used for the selections were pre-blocked with ChemiBLOCKER(Chemicon, Temecula, Calif., USA). HuCAL GOLD® phage were blocked withChemiBLOCKER (+0.05% Tween20) and pre-adsorbed twice on M-280Streptavidin Dynabeads® (Dynal Biotech, Oslo, Norway). Pre-blocked phageand biotinylated OPN protein or peptide antigen were incubated in a 2 mLtube for 2 hours at room temperature on a rotator. For the firstselection round, 100 nM of biotinylated antigen concentration was usedfor bead coupling. The second and third panning rounds were performedusing 10 nM biotinylated antigens.

Pre-adsorbed Streptavidin Dynabeads® were added to the phage-antigensolution and incubated further for 10 minutes at room temperature on arotator. A magnetic particle separator, MPC-E (Dynal Biotech, Oslo,Norway), was used to separate phage bound to the captured antigen. Thebeads were washed several times with PBST (PBS, 0.05% Tween 20),followed by several washing steps with PBS. The washing stringency wasincreased with every panning round. PBS was removed after the lastwashing step before continuing with elution. Elution and further stepswere performed as described previously for the solid phase panning.

Subcloning and Microexpression of Selected Fab Fragments

To facilitate rapid expression of soluble Fab, the Fab encoding insertsof the selected HuCAL GOLD® phage were subcloned via XbaI and EcoRI intothe expression vector pMORPH®X9_MH. After transformation of theexpression plasmids into E. coli TG1 F-cells chloramphenicol-resistantsingle clones were picked into the wells of a sterile 384-wellmicrotiter plate pre-filled with 2×YT medium (supplemented with 34 μg/mLchloramphenicol and 1% glucose) and grown overnight at 37° C. Theseplates were regarded as master plates. Before storage of the masterplates at −80° C., the E. coli TG1 F-cultures were inoculated into new,sterile 384-well microtiter plates pre-filled with 40 μL 2×YT mediumsupplemented with 34 μg/mL chloramphenicol and 0.1% glucose per well.The microtiter plates were incubated at 30° C. shaking at 400 rpm on amicroplate shaker until the cultures were slightly turbid (˜2 to 4hours) with an OD600 of ˜0.5. These plates were regarded as expressionplates, and 10 μL 2×YT medium supplemented with 34 μg/mL chloramphenicoland 5 mM IPTG was added per well (end concentration 1 mM IPTG), themicrotiter plates were sealed with a gas-permeable tape, and incubatedovernight at 30° C. shaking at 400 rpm.

Generation of whole cell lysates (BEL extracts): To each well of theexpression plates, 15 μL BEL buffer was added and incubated for 1 hourat 22° C. on a microtiter plate shaker (400 rpm). BEL buffer: 24.7 g/Lboric acid, 18.7 g NaCl/L, 1.49 g EDTA/I, pH 8.0 supplemented with 2.5mg/mL lysozyme.

Expression and Purification of HuCAL-Fab Antibodies in E. Coli

Expression of Fab fragments encoded by pMORPHX9_FH in TG-1 F-cells wascarried out in shaker flask cultures with 1 L of 2×TY mediumsupplemented with 34 μg/mL chloramphenicol. After induction with 0.5 mMIPTG, cells were grown at 30° C. for 20 hours. Whole cell lysis(Lysozyme) of cell pellets were prepared and Fab fragments isolated byHT-IMAC-purification. The apparent molecular weights were determined bysize exclusion chromatography (SEC) with calibration standards.Concentrations were determined by UV-spectrophotometry.

Example 2 Screening of OPN Positive Clones

OPN positive clones were further identified by screening the clonesgenerated in Example 1 for antigen binding using the ELISA assay methodsas described below.

Screening on Directly Coated OPN

Primary and secondary screening was performed using hOPN and mOPNprotein as well as SSP1 peptides. hOPN was used for overnight coating ofMaxisorp® microtiter plates at 4° C. at a concentration of 12.5 μg/mL(diluted in PBS), mOPN was coated at a concentration of 5 μg/mL.

After overnight incubation, coated plates were washed twice with PBST(PBS/0.05% Tween20) and blocked with 5% MPBST (5% milkpowder in PBST)for 1 hour at room temperature on a microplate shaker. The plates werewashed twice with PBST before primary antibodies were added (crudeextracts of microexpressed HuCAL® Fabs, purified HuCAL® Fabs, anti-OPNmonoclonal control antibody AKm2A1, 1:200, Santa Cruz #SC-21742). Theplates containing the primary antibodies were incubated for 1 hour atroom temperature on a microplate shaker. The plates were washed twicewith PBST and for the detection of HuCAL® Fabs the secondary antibody(Goat anti-human F(ab)₂—Fragment specific—AP labeled, Jackson Cat. No.109-055-097) was added, diluted 1:5000 in 0.5% MPBST. The platecontaining the secondary antibodies was incubated for 1 hour at roomtemperature on a microplate shaker. The wells were washed five timeswith TBST (TBS/0.05% Tween20), Attophos (AttoPhos Substrate Set, Roche,#11681982001) was added (diluted 1:10 in water) and fluorescenceemission at 535 nm was recorded with excitation at 430 nm.

Capture Screening Using Biotinylated OPN Protein

Maxisorp (Nunc, Rochester, N.Y., USA) 384 well plates were coated with20 μL/well NeutrAvidin™ biotin binding protein (Pierce, Cat. No. #31000)at a final concentration of 10 μg/mL diluted in PBS, pH 7.4 byincubation over night at 4° C. and 450 rpm on a microplate shaker.

The following day, the plates were washed two times using TBST(TBS/0.05% Tween20) and blocked for 1 hour using 90 μL/well Superblocksolution (Pierce, Cat. No. #37545). Blocked plates were washed threetimes using TBST followed by incubation for 2 hours of 10 μL/wellbiotinylated human or mouse OPN protein (2 μg/mL). Plates were washedthree times using TBST followed by blocking for 1 hour by incubation of90 mL/well 10% BSA in TBS. Plates were finally washed five times usingTBST before 40 μL/well BEL extract was incubated for 1.5 hours at roomtemperature. Subsequently HuCAL® Fab fragments were allowed to bind tocaptured biotinylated antigen OPN. The plates were washed twice withPBST and for the detection of HuCAL® Fabs the secondary antibody (Goatanti-human F(ab)₂—Fragment specific—AP labeled, Jackson Cat. No.#109-055-097) was added, diluted 1:5000 in 0.5% MPBST. The platecontaining the secondary antibodies was incubated for 1 hour at roomtemperature on a microplate shaker. The wells were washed five timeswith TBST, Attophos (AttoPhos Substrate Set, Roche, #11681982001) wasadded (diluted 1:10 in water) and fluorescence emission at 535 nm wasrecorded with excitation at 430 nm.

The EC₅₀ values (determined as described above) for twelve selected Fabsare shown below in Table 2.

TABLE 2 Summary of ELISA EC₅₀ Values for Twelve Selected Fabs ProteinELISA Peptide ELISA hOPN mOPN hOPN mOPN Fab EC₅₀ Std EC₅₀ Std EC₅₀ StdEC₅₀ Std 6453 1.3 0.5 2.8 1.6 no binding no binding 6454 1.5 0.8 393.0179.6 no binding no binding 6455 0.7 0.4 n.d. n.d. no binding no binding6989 1.8 2.4 3.7 3.5 0.7 0.4 0.5 0.4 6990 8.3 7.9 3.3 2.1 1.2 1.1 0.40.3 6991 3.4 3.3 327.2 214.1 0.3 0.1 49.0 37.00.5 6992 0.9 0.6 4.5 3.13.9 5.0 1.0 0.1 6993 1.2 0.8 0.9 0.7 0.4 0.3 0.3 7201 10.5 8.1 15.0 14.1no binding no binding 7202 242.0 196.6 16.8 10.7 no binding no binding7203 1.7 2.0 12.2 9.8 1.2 n.d. 2.2 1.3 7212 5.2 0.8 9.8 2.1 no bindingno binding n.d. = not determined; Std = standard deviation

The EC₅₀ values (determined as described above) for seven of theselected IgGs (seven selected Fabs were converted to full length humanIgG2 antibodies as described in Example 4) are shown below in Table 3.

TABLE 3 Summary of ELISA EC₅₀ Values for Seven Selected IgGs ProteinELISA Peptide ELISA hOPN mOPN hOPN mOPN Fab EC₅₀ Std EC₅₀ Std EC₅₀ StdEC₅₀ Std 6454 1.7 0.4 248.7 45.4 no binding no binding 6455 1.6 0.2 31.41.4 no binding no binding 6990 2.7 0.5 1.6 0.5 1.1 0.5 1.1 0.3 6991 2.70.6 8.5 2.3 0.6 0.4 1.9 1.3 6993 7.1 6.9 2.0 1.5 1.8 1.5 0.9 0.8 72023.7 1.0 2.5 0.8 no binding no binding 7212 3.7 0.4 5.3 0.1 no binding nobinding Std = standard deviation

Example 3 Characterization of HuCAL GOLD® Fabs and IgGs

Selected HuCAL GOLD® Fabs and IgGs were further characterized usingseveral assays as described below, as well as with the ELISA techniquesas described in Example 2.

Cell Adhesion Assay

HuCAL GOLD® Fabs and IgGs were tested for their ability to inhibit OPNmediated adhesion of metastatic breast cancer cell line MDA-MB 435 (ATCC#HTB-129) in a Mn²⁺-dependent, as well as Mn²⁺-independent setup. AMaxisorp™ plate (Nunc Cat. No. #43711) was coated overnight at 4° C.with 50 μL/well of 1 μg/mL hOPN diluted in PBS+ (PBS supplemented with100 μg/mL CaCl₂, 100 μg/mL MgCl₂) with or without 0.5 mM MnCl₂. Thefollowing day, plates were washed twice with PBS+ and blocked with TBScontaining 10% BSA for 2 hours at 37° C., 5% CO₂. After blocking, theplates were washed twice with PBS+ and once with adhesion buffer II(HBSS, Gibco #14025-100; 50 nM HEPES Buffer Solution, Gibco #15630-056;1 mg/mL BSA; 1 mM MnCl₂). HuCAL GOLD® Fabs or IgGs were diluted inadhesion buffer II to the indicated concentrations and 50 μL/well wereadded. Plates were incubated for 1 hour at 37° C., 5% CO₂.

MDA-MB453 cells were detached using Accutase (PAA Laboratories#L11-007). 1×10⁶ cells/mL were resuspended in adhesion buffer I (HBSS,Gibco #14025-100; 50 nM HEPES Buffer Solution, Gibco #15630-056; 1 mg/mLBSA) and incubated with calcein AM (1 μg/mL/10⁶ cells, Invitrogen#C3099) for 45 minutes at 37° C., 5% CO₂. Cells were centrifuged andresuspended in adhesion buffer II at a concentration of 1×10⁶ cells/mL.50 μL of cells (1×10⁵ cells/well) were added to antibody containingwells and incubated for 90 minutes at 37° C., 5% CO₂. Adhesion wasstopped by gently washing the wells 5 times with adhesion buffer IIfollowed by washing two times with PBS+ leaving 100 μL PBS/well afterthe last washing step. Fluorescence emission at 535 nm was recorded withexcitation at 485 nm.

Cell adhesion data (determined as described above) for twelve selectedFabs are shown below in Table 4. For two of the Fabs (7201 and 7202), noadhesion activity was observed. Fab 7212 was not evaluated. For theMn2+-independent adhesion assay, IC50 values could not be determined. Aninhibitory effect, however, was observed (indicated by a (+)) for allFabs except 7201 and 7202.

TABLE 4 Summary of Cell Adhesion Data for Twelve Selected Fabs AdhesionAssay Mn²⁺-dependent Adhesion Assay hOPN Mn²⁺-independent Fab IC₅₀ (nM)Std hOPN 6453 117.3 13.7 (+) 6454 95.1 42.1 (+) 6455 131.4 45.5 (+) 698966.9 27.4 (+) 6990 156.8 47.3 (+) 6991 264.3 118.7 (+) 6992 98.5 40.1(+) 6993 29.8 22.6 (+) 7201 No inhibition No inhibition 7202 Noinhibition No inhibition 7203 56.1 15.6 (+) 7212 Not evaluated (+) Std =standard deviation

Cell adhesion data (determined as described above) for seven selectedIgGs (seven selected Fabs were converted to full length human IgG2antibodies as described in Example 4) are shown below in Table 5. Fortwo of the IgGs (6455 and 7202), no adhesion activity was observed. Forthe Mn²⁺-independent adhesion assay, EC₅₀ values could not bedetermined. An inhibitory effect, however, was observed (indicated by a(+)) for all Fabs except 6455 and 7202.

TABLE 5 Summary of Cell Adhesion Data for Seven Selected IgGs AdhesionAssay Mn²⁺-dependent Adhesion Assay hOPN Mn²⁺-independent IgGs IC₅₀ (nM)Std hOPN 6454 1.3 1.4 (+) 6455 No inhibition No inhibition 6990 0.4 0.4(+) 6991 0.6 0.1 (+) 6993 0.4 0.4 (+) 7202 No inhibition No inhibition7212 9.7 4.0 (+) Std = standard deviation

Affinity Determination Using Solution Equilibrium Titration (SET)

For K_(D) determination by solution equilibrium titration (SET), monomerfractions of antibody protein were used (at least 90% monomer content,analyzed by analytical SEC; Superdex75 (Amersham Pharmacia) for Fab, orTosoh G3000SWXL (Tosoh Bioscience) for IgG, respectively). Affinitydetermination in solution was basically performed as described in theliterature (Friguet et al., J. Immunol. Methods 77:305 (1985)). In orderto improve the sensitivity and accuracy of the SET method, it wastransferred from classical ELISA to ECL based technology (Haenel et al.,Anal. Biochem. 339:182-184 (2005)). 1 mg/mL goat-anti-human (Fab)₂fragment specific antibodies (Dianova) were labeled with MSD Sulfo-TAG™NHS-Ester (Meso Scale Discovery, Gaithersburg, Md., USA) according tothe manufacturer's instructions. The experiments were carried out inpolypropylene microtiter plates and PBS, pH 7.4, with 0.5% BSA and 0.02%Tween 20 as assay buffer. Unlabeled osteopontin was diluted in a 2nseries, starting with a concentration at least 10 times higher than theexpected K_(D). Wells without antigen were used to determine B_(max)values; wells with assay buffer were used to determine background. Afteraddition of e.g. 25 pM Fab (final concentration in 60 μL final volume),the mixture was incubated over night at room temperature. The appliedFab concentration was similar to or below the expected K_(D).

Streptavidin coated MSD plates were blocked over night with 3% BSA inPBS (50 μL/well), subsequently the blocking solution was discarded andthe plates were coated with 0.2 pg/mL biotinylated osteopontin in assaybuffer (30 μL/well) for 1 hour. After washing the coated MSD plates withassay buffer, the equilibrated samples were transferred to those plates(30 μL/well) and incubated for 20 minutes. After washing, 30 μL/well ofthe MSD Sulfo-tag labeled detection antibody (goat anti-human (Fab)₂) ina final dilution of 1:1000 was added to the MSD plate and incubated for30 minutes on an Eppendorf shaker (700 rpm).

After washing the plate and adding 30 μL/well MSD Read Buffer T withsurfactant, electro-chemiluminescence signals were detected using aSector Imager 6000 (Meso Scale Discovery, Gaithersburg, Md., USA).

The data was evaluated with XLfit (IDBS) software applying customizedfitting models. For K_(D) determination of Fab molecules, a fit modelwas used according to Haenel et al., Anal. Biochem. 339:182-184 (2005).

A similar protocol was applied to determine K_(D) values for IgGmolecules, with the following differences: instead of Fab molecules,whole IgG molecules were added to the dilution series of antigen, andequilibrated over night at room temperature. Subsequently, the sampleswere treated as described above. KD values for IgG molecules were thendetermined using a fitting model that was modified according to Piehleret al., J. Immunol. Methods 201:189-192 (1997).

Biacore K_(D) Determination on Directly Coated Antigen

For K_(D) determination, monomeric Fab fractions (at least 90% monomercontent, analyzed by analytical SEC; Superdex75, Amersham Pharmacia)were used as analyte. Binding to immobilized antigen was analyzed usingthe BIAcore3000 instrument (Biacore, Sweden).

For antigen immobilization, two alternative strategies were used: in thecase of human OPN, biotinylated human OPN was bound to a Streptavidincoated sensor chip (Biacore, Sweden) to a binding level of approximately300 RU. The reference flow cell was coated with a similar amount ofbiotinylated HSA. In the case of murine OPN, the antigen was immobilizedcovalently using standard EDC-NHS amine coupling chemistry. CM5 chips(Biacore, Sweden) were coated with murine OPN in 10 mM acetate buffer,pH 3.5 to a level of 400 RU. For the reference flow cell, a respectiveamount of HSA was used. Regeneration was accomplished with twoinjections of 10 mM Gly/HCl, pH 1.5 (5 μL) and 50 mM phosphoric acid (5μL), respectively. Kinetic measurements were done in Dulbeccos PBS, pH7.4 (Gibco) with 0.05% Tween 20 at a flow rate of 20 μL/min using a 2nserial dilution row of Fab samples. The Fab concentrations ranged from15.6 to 500 nM. The injection time for each concentration was 1 minute,and the dissociation time was set to a minimum of 3 minutes. A blankinjection of running buffer was used for double referencing. Allsensorgrams were fitted globally using BIA evaluation software 3.2(Biacore, Sweden), to determine association and dissociation rateconstants (k_(on) and k_(off)), which were used to calculate theaffinity (K_(D)=k_(off)/k_(on)).

Biacore affinity and/or SET affinity K_(D) data (determined as describedabove) for twelve selected Fabs are shown below in Table 6.

TABLE 6 Summary of Affinities for Twelve Selected Fabs Biacore AffinityKD (nM) SET Affinity (nM) Fab hOPN mOPN hOPN 6453 373 9 n.d. 6454 16 100.4 6455 29 6 11 6989 120 2065 n.d. 6990 140 2272 35 6991 300 1388 36992 100 2600 n.d. 6993 70 3985 0.2 7201 20 3300 n.d. 7202 220 9900 67203 210 1628 3 7212 n.d. 2463 13 n.d. = not determined

Example 4 Conversion of Fabs to IgG

In order to express full length IgG, variable domain fragments of heavy(VH) and light chains (VL) were subcloned from Fab expression vectorsinto appropriate pMorph®_hlg vectors for human IgG2a. Variable domainswere amplified via PCR using appropriate oligonucleotides (Fab_HC_for:5′-CCT ACC GTT CGT CTT CAC CCC TG-3′ (SEQ ID NO:70); Fab_LC_for: 5′-GGCACT GGC TGG TTT CGC TAC-3′ (SEQ ID NO:71); Fab_HC_rev: 5′-CTC GGA GCCAGC GGA AAC AC-3′ (SEQ ID NO:72); Fab_kappa_rev: 5′-CGG AAA AAT AAA CACGCT CGG A-3′ (SEQ ID NO:73); Fab_lambda_rev: 5′-GCT CAC ACT CGG TGC GGCTTT C-3′ (SEQ ID NO:74)) followed by digestion with MfeI, BlpI (VH),EcoRV, HpaI (VLlambda) or EcoRV, BsiWI (VKappa), respectively. Fragmentswere used for subcloning into pMorph®_hlg2κ_(—)1, pMorph®_hlg2λ_(—)1 orpMorph®_hlgG2.

Transient Expression and Purification of Human IgG2

Transient expression of full length human IgG2 was performed in HKB11cells, which were transfected with IgG2 heavy and light chain expressionvectors. Cell culture supernatant was harvested either three days aftertransfection or seven days after transfection and upscaling to 3-foldtransfection volume, respectively. Supernatant was cleared bycentrifugation.

After filtration (0.22 μm or 0.45 μm), the supernatant was subjected tostandard protein A affinity chromatography (MabSelect SURE, GE).Proteins were eluted at pH 3 and neutralized in 3 M TRIS, pH 8. Furtherdownstream processing involved buffer exchange to 1× Dubleccos' PBS(Invitrogen) and sterile filtration (0.2 μm; Millipore or Sartorius).Purity of IgG2 was analyzed under denaturing, reducing and denaturing,non-reducing conditions in SDS-PAGE or by capillary electrophoresis.HP-SEC was performed to analyze IgG2 preparations in their native state.

The procedures described above in Examples 1 to 4 were used to produceseveral fully human anti-OPN IgG₂ antibodies, including antibodiesdesignated as “MOR-6990” (or 6990), “MOR-6991” (or 6991), and “MOR-6993”(or 6993), which are described herein.

Example 5 Structural Characterization of Human Antibodies MOR-6990,MOR-6991, and MOR-6993

The cDNA sequences encoding the heavy and light chain variable regionsof the MOR-6990, MOR-6991, and MOR-6993 monoclonal antibodies wereobtained from the respective hybridomas using standard PCR techniquesand were sequenced using standard DNA sequencing techniques.

The nucleotide and amino acid sequences of the heavy chain variableregion of 6990 are shown in FIGS. 1A and 1B and in SEQ ID NOs: 9 and 7,respectively. The nucleotide and amino acid sequences of the light chainvariable region of 6990 are shown in FIGS. 1C and 1D and in SEQ ID NOs:10 and 8, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 6991 are shown in FIGS. 1E and 1F and in SEQ ID NOs: 23 and21, respectively. The nucleotide and amino acid sequences of the lightchain variable region of 6990 are shown in FIGS. 1G and 1H and in SEQ IDNOs: 24 and 22, respectively.

The nucleotide and amino acid sequences of the heavy chain variableregion of 6993 are shown in FIGS. 1I and 1J and in SEQ ID NOs: 37 and35, respectively. The nucleotide and amino acid sequences of the lightchain variable region of 6990 are shown in FIGS. 1K and 1L and in SEQ IDNOs: 38 and 36, respectively.

As described in Example 4, the 6990, 6991, and 6993 human antibodies areof isotype IgG2. The nucleotide and amino acid sequences of thefull-length heavy chain for 6990 are shown in FIGS. 2A and 2B, and inSEQ ID NOs: 13 and 11, respectively. The nucleotide and amino acidsequences of the full-length light chain for 6990 are shown in FIGS. 2Cand 2D, and in SEQ ID NOs: 14 and 12, respectively.

The nucleotide and amino acid sequences of the full-length heavy chainfor 6991 are shown in FIGS. 2E and 2F, and in SEQ ID NO: 27 and 25,respectively. The nucleotide and amino acid sequences of the full-lengthlight chain for 6991 are shown in FIGS. 2G and 2H, and in SEQ ID NO: 28and 26, respectively.

The nucleotide and amino acid sequences of the full-length heavy chainfor 6993 are shown in FIGS. 2I and 2J, and in SEQ ID NO: 41 and 39,respectively. The nucleotide and amino acid sequences of the full-lengthlight chain for 6993 are shown in FIGS. 2K and 2L, and in SEQ ID NO: 42and 40, respectively.

Comparison of the 6990 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 6990 heavy chain utilizes a V_(H) segment from human germline V_(H)3-23, a D segment from the human germline 3-22, and a JH segment fromhuman germline JH 4a. Further analysis of the 6990 V_(H) sequence usingthe Kabat system of CDR region determination led to the delineation ofthe heavy chain CDR1, CDR2 and CDR3 regions as shown in FIG. 1B, and inSEQ ID NOs: 1, 2 and 3, respectively.

Comparison of the 6990 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 6990 light chain utilizes a V_(L) segment from human germline λ3 anda JL segment from human germline JL 3b. Further analysis of the 6990V_(L) sequence using the Kabat system of CDR region determination led tothe delineation of the light chain CDR1, CDR2 and CDR3 regions as shownin FIG. 1D, and in SEQ ID NOs: 4, 5 and 6, respectively.

Comparison of the 6991 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 6991 heavy chain utilizes a V_(H) segment from human germline V_(H)3-23, a D segment from the human germline 2-21, and a JH segment fromhuman germline JH 4a. Further analysis of the 6991 V_(H) sequence usingthe Kabat system of CDR region determination led to the delineation ofthe heavy chain CDR1, CDR2 and CDR3 regions as shown in FIG. 1F, and inSEQ ID NOs: 15, 16 and 17, respectively.

Comparison of the 6991 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 6991 light chain utilizes a V_(L) segment from human germline λ1-13and a JL segment from human germline JL 3b. Further analysis of the 6991V_(L) sequence using the Kabat system of CDR region determination led tothe delineation of the light chain CDR1, CDR2 and CDR3 regions as shownin FIG. 1H, and in SEQ ID NOs: 18, 19 and 20, respectively.

Comparison of the 6993 heavy chain immunoglobulin sequence to the knownhuman germline immunoglobulin heavy chain sequences demonstrated thatthe 6993 heavy chain utilizes a V_(H) segment from human germline V_(H)3-23, a D segment from the human germline 3-10, and a JH segment fromhuman germline JH 4a. Further analysis of the 6993 V_(H) sequence usingthe Kabat system of CDR region determination led to the delineation ofthe heavy chain CDR1, CDR2 and CDR3 regions as shown in FIG. 1J, and inSEQ ID NOs: 29, 30 and 31, respectively.

Comparison of the 6993 light chain immunoglobulin sequence to the knownhuman germline immunoglobulin light chain sequences demonstrated thatthe 6993 light chain utilizes a V_(L) segment from human germline λ3 anda JL segment from human germline JL 3b. Further analysis of the 6993V_(L) sequence using the Kabat system of CDR region determination led tothe delineation of the light chain CDR1, CDR2 and CDR3 regions as shownin FIG. 1 L, and in SEQ ID NOs: 32, 33 and 34, respectively.

Example 6 Germlined Versions of Human Antibodies MOR-6990, MOR-6991, andMOR-6993

In order to minimize immunogenicity of the 6990, 6991, and 6993antibodies, several mutations were returned to germ line sequence, asfollows. A germlined version of 6990 (6990-GL) was prepared by returningtwo amino acids in the FR1 region of the heavy variable chain to germline sequence. The two amino acid residues (and corresponding nucleicacid codons) that were changed in the heavy chain variable region can beseen in FIGS. 1M and 1N, where the mutated residues are indicated byboxing. In the light chain variable region of 6990, five amino acids inthe FR1 region and one in the FR3 region were returned to germ linesequence. The six amino amino acids (and corresponding nucleic acidcodons) that were changed in the light chain variable region can be seenin FIGS. 1O and 1P, where the mutated residues are indicated by boxing.

A germlined version of 6991 (6991-GL) was prepared by returning twoamino acids in the FR1 region of the heavy variable chain to germ linesequence. The two amino acid residues (and corresponding nucleic acidcodons) that were changed in the heavy chain variable region can be seenin FIGS. 1Q and 1R, where the mutated residues are indicated by boxing.In the light chain variable region of 6991, two amino acids in the FR1region and one in the FR3 region were returned to germ line sequence.The three amino amino acids (and corresponding nucleic acid codons) thatwere changed in the light chain variable region can be seen in FIGS. 1Sand 1T, where the mutated residues are indicated by boxing.

A germlined version of 6993 (6993-GL) was prepared by returning twoamino acids in the FR1 region of the heavy variable chain to germ linesequence. The two amino acid residues (and corresponding nucleic acidcodons) that were changed in the heavy chain variable region can be seenin FIGS. 1U and 1V, where the mutated residues are indicated by boxing.In the light chain variable region of 6993, five amino acids in the FR1region, one amino acid in the FR2 region, and one in the FR3 region werereturned to germ line sequence. The seven amino amino acids (andcorresponding nucleic acid codons) that were changed in the light chainvariable region can be seen in FIGS. 1W and 1X, where the mutatedresidues are indicated by boxing.

Example 7 Preparation of Mutant to Improve Solubility

In order to improve the solubility of 6993-GL, a point mutation wasintroduced in the FR2 region of the light chain variable region (V44K).This point mutation (and the corresponding nucleic acid codon) can beseen in FIGS. 1Y and 1Z, where the point mutation is indicated by boldtext.

Example 8 Characterization of Binding Avidity of Osteopontin HumanMonoclonal Antibodies (MOR6990, MOR6991 and MOR6993)

Binding avidity of monoclonal antibodies to Osteopontin was determinedusing Biacore analysis (General Electric Healthcare, Biacore 3000). Toobtain nominal avidity measurements, human Osteopontin (R&D Systems,1433-OP-050/CF) and mouse Osteopontin (R&D Systems, 441-OP-050/CF) wereimmobilized on a biosensor chip using standard amine coupling andvarious concentrations of monoclonal antibodies (MOR6990, MOR6991 andMOR6993) were flowed across the surface at 25.0° C. in 10 mM HEPES pH7.4, 150 mM NaCl, 0.005% P20. The binding data were fit globally to asimple one-to-one binding model. The results are shown in Table 7.

TABLE 7 Summary of Avidities for MOR6990, MOR6991, and MOR6993 MouseOsteopontin Human Osteopontin k_(a) (1/Ms) k_(d) (1/s) K_(D) (Avidity)k_(a) (1/Ms) k_(d) (1/s) K_(D) (Avidity) MOR6990 1.83E+05 8.64E−04  4.7nM 9.69E+04 2.03E−03  20.9 nM MOR6991 N/A N/A N/A 4.91E+05 9.33E−03 19.0 nM MOR6993 6.64E+06 4.96E−03 746.7 pM 6.14E+06 4.62E−03 752.0 pMN/A = No detectable binding of MOR6991 to mouse Osteopontin

Example 9 Inhibition of In Vivo Tumor Growth and Metastasis by MOR-6993

The following study demonstrated the anti-metastatic efficacy ofMOR-6993 in a preclinical model of breast cancer.

MDA-MB-435-Luc is a human breast cancer cell line transfected with theluciferase gene. When implanted into the mouse mammary fat pad (MFP), itinduces formation of tumors that can be measured by bioluminescenceimaging (BLI).

In this study, MDA-MB-435-Luc cells were injected (3×10⁶ per animal) inthe mammary fat pad of immuno-compromised SCID BALB/c mice that hadreceived a pre-dose of MOR-6993 antibody (30 mg/kg, n=10 per group) 24hours before implantation. Following implantation, animals were dosedonce a week subcutaneously with 6993 (10 mg/kg) for 6 weeks.Bioluminescence of individual animals was measured once a week for 10weeks. Tumors were surgically removed on day 40 from implant, andanimals were monitored for additional 50 days for appearance ofmetastasis as well as overall survival.

Dosing with 6993 resulted in significant TGI (tumor growth inhibition),30% tumor weight reduction as shown in FIG. 5A. In addition, thetreatment prevented or delayed the appearance of metastases (FIG. 5B)and improved overall survival. RNA analysis of the tumors upon removalshowed that treatment with 6993 induced a decrease in OPN mRNAexpression.

Example 10 In Vivo Neutralization of Circulating Osteopontin

The following study demonstrated neutralization of both endogenous mouseand tumor-produced Osteopontin by Mor-6990 and Mor-6993.

MDA-MB-435-Hal/Luc cells were injected (3×10⁶ per animal) in the mammaryfat pad of immuno-compromised SCID BALB/c mice to induce tumorformation. When the tumors reached 500 mm³, animals were randomized anddosed intravenously with Mor-6990 or Mor-6993 at 25 mg/kg. Blood wascollected at 1 and 24 hr post-dose. Total mouse and human Osteopontin inplasma were analyzed using a commercial ELISA kit (R&D systems). Foranalysis of the free Osteopontin, plasma was treated overnight withProtein G magnetic beads to remove Osteopontin bound to the IgGs. FreeOsteopontin levels were determined using a commercial ELISA kit (R&Dsystems). The results demonstrated that both Mor-6990 and Mor-6993effectively neutralized both mouse (FIG. 6A) and human (FIG. 6B) plasmaOsteopontin.

Example 11 ELISA EC₅₀ Values and Binding Affinities of MOR-10475 Fabs

Protein ELISA EC₅₀ values, peptide ELISA EC₅₀ values, and Biacore K_(D)values were determined for MOR-10475 Fabs using both human and mouseosteopontin in a similar manner to procedures described in Examples 2and 3, and the results are shown in Table 8.

TABLE 8 ELISA EC₅₀ and Biacore K_(D) values of MOR-10475 Protein ELISAPeptide ELISA Biacore hOPN mOPN hOPN mOPN hOPN mOPN Fab EC₅₀ (nM) EC₅₀(nM) EC₅₀ (nM) EC₅₀ (nM) K_(D) (nM) K_(D) (nM) 10475 0.6 0.6 0.7 0.7 8041

SUMMARY OF SEQUENCE LISTING (H-CDR1=heavy chain CDR1; L-CDR1=light chainCDR1, etc; 6990=antibody MOR-6990 as described herein; 6991=antibodyMOR-6991 as described herein; 6993=antibody MOR-6993 as describedherein; VH=variable heavy region; VL=variable light region;Heavy=full-length heavy chain; Light=full-length light chain;6990-GL=germlined version of MOR-6990, as described herein;6991-GL=germlined version of MOR-6991, as described herein;6993-GL=germlined version of MOR-6993, as described herein;6993-GL-V44K=germlined version of MOR-6993, including the V44K pointmutation, as described herein; a.a.=amino acid; n.a.=nucleic acid)

SEQ ID NO: Description 1 H-CDR1 a.a. 6990 2 H-CDR2 a.a. 6990 3 H-CDR3a.a. 6990 4 L-CDR1 a.a. 6990 5 L-CDR2 a.a. 6990 6 L-CDR3 a.a. 6990 7V_(H) a.a. 6990 8 V_(L) a.a. 6990 9 V_(H) n.a. 6990 10 V_(L) n.a. 699011 Heavy a.a. 6990 12 Light a.a. 6990 13 Heavy n.a. 6990 14 Light n.a.6990 15 H-CDR1 a.a. 6991 16 H-CDR2 a.a. 6991 17 H-CDR3 a.a. 6991 18L-CDR1 a.a. 6991 19 L-CDR2 a.a. 6991 20 L-CDR3 a.a. 6991 21 V_(H) a.a.6991 22 V_(L) a.a. 6991 23 V_(H) n.a. 6991 24 V_(L) n.a. 6991 25 Heavya.a. 6991 26 Light a.a. 6991 27 Heavy n.a. 6991 28 Light n.a. 6991 29H-CDR1 a.a. 6993 30 H-CDR2 a.a. 6993 31 H-CDR3 a.a. 6993 32 L-CDR1 a.a.6993 33 L-CDR2 a.a. 6993 34 L-CDR3 a.a. 6993 35 V_(H) a.a. 6993 36 V_(L)a.a. 6993 37 V_(H) n.a. 6993 38 V_(L) n.a. 6993 39 Heavy a.a. 6993 40Light a.a. 6993 41 Heavy n.a. 6993 42 Light n.a. 6993 43 Full-lengthhuman OPN a.a. isoform b 44 V_(H) a.a. 6990-GL 45 V_(H) n.a. 6990-GL 46V_(L) a.a. 6990-GL 47 V_(L) n.a. 6990-GL 48 V_(H) a.a. 6991-GL 49 V_(H)n.a. 6991-GL 50 V_(L) a.a. 6991-GL 51 V_(L) n.a. 6991-GL 52 V_(H) a.a.6993-GL 53 V_(H) n.a. 6993-GL 54 V_(L) a.a. 6993-GL 55 V_(L) n.a.6993-GL 56 V_(L) a.a. 6993-GL-V44K 57 V_(L) n.a. 6993-GL-V44K 58 Heavy6990-GL a.a. 59 Light 6990-GL a.a. 60 Heavy 6990-GL n.a. 61 Light6990-GL n.a. 62 Heavy 6991-GL a.a. 63 Light 6991-GL a.a. 64 Heavy6991-GL n.a. 65 Light 6991-GL n.a. 66 Heavy 6993-GL a.a. 67 Light6993-GL a.a. 68 Heavy 6993-GL n.a. 69 Light 6993-GL n.a. 70 Fab heavychain forward primer 71 Fab light chain forward primer 72 Fab heavychain reverse primer 73 Fab kappa reverse primer 74 Fab lambda reverseprimer 75 L-CDR3 a.a. MOR10475 76 V_(L) a.a. MOR10475 77 V_(L) n.a.MOR10475 78 Light a.a. MOR10475

1. An isolated antibody, or antigen-binding portion thereof, thatspecifically binds to osteopontin, wherein said antibody orantigen-binding portion comprises: (a) an H-CDR1 as set forth in SEQ IDNO:1, an H-CDR2 as set forth in SEQ ID NO:2, an H-CDR3 as set forth inSEQ ID NO:3, an L-CDR1 as set forth in SEQ ID NO:4, an L-CDR2 as setforth in SEQ ID NO:5, and an L-CDR3 as set forth in SEQ ID NO:6; (b) anH-CDR1 as set forth in SEQ ID NO:15, an H-CDR2 as set forth in SEQ IDNO:16, an H-CDR3 as set forth in SEQ ID NO:17, an L-CDR1 as set forth inSEQ ID NO:18, an L-CDR2 as set forth in SEQ ID NO:19, and an L-CDR3 asset forth in SEQ ID NO:20; (c) an H-CDR1 as set forth in SEQ ID NO:29,an H-CDR2 as set forth in SEQ ID NO:30, an H-CDR3 as set forth in SEQ IDNO:31, an L-CDR1 as set forth in SEQ ID NO:32, an L-CDR2 as set forth inSEQ ID NO:33, and an L-CDR3 as set forth in SEQ ID NO:34; or (d) anH-CDR1 as set forth in SEQ ID NO:1, an H-CDR2 as set forth in SEQ IDNO:2, an H-CDR3 as set forth in SEQ ID NO:3, an L-CDR1 as set forth inSEQ ID NO:4, an L-CDR2 as set forth in SEQ ID NO:5, and an L-CDR3 as setforth in SEQ ID NO:75.
 2. The antibody according to claim 1, which is anIgG1 or IgG2.
 3. The antibody according to claim 1, which is a human,humanized, or chimeric antibody.
 4. The antibody according to claim 3,which is a synthetic human antibody.
 5. The antigen-binding portionaccording to claim 1, which is a Fab or scFv antibody fragment.
 6. Anucleic acid encoding the antibody or antigen-binding portion accordingto claim
 1. 7. A vector comprising the nucleic acid according to claim6.
 8. A host cell comprising the vector according to claim
 7. 9. Thehost cell according to claim 8, wherein said cell is bacterial.
 10. Thehost cell according to claim 8, wherein said cell is mammalian.
 11. Apharmaceutical composition comprising an antibody or antigen-bindingportion according to claim 1 and a pharmaceutically acceptable carrieror excipient.
 12. A method for treating abnormal cell growth comprisingadministering to a subject in need thereof an effective amount of apharmaceutical composition according to claim
 11. 13. A method ofreducing tumor cell metastasis in a subject, comprising administering tosaid subject an effective amount of a pharmaceutical compositionaccording to claim
 11. 14. A method of preparing an anti-ostepontinantibody, or antigen-binding portion thereof, comprising expressing theantibody or antigen-binding portion in a host cell according to claim 8.15. An isolated antibody, or antigen-binding portion thereof, thatspecifically binds to osteopontin, wherein said antibody orantigen-binding portion comprises a V_(H) chain amino acid sequence asset forth in SEQ ID NO:7, SEQ ID NO:21, SEQ ID NO:35, SEQ ID NO:44, SEQID NO:48, or SEQ ID NO:52.
 16. The antibody or antigen-binding portionaccording to claim 15, wherein said antibody or antigen-binding portionfurther comprises a V_(L) chain amino acid sequence as set forth in SEQID NO:8, SEQ ID NO:22, SEQ ID NO:36, SEQ ID NO:46, SEQ ID NO:50, SEQ IDNO:54, or SEQ ID NO:76.
 17. An isolated antibody that specifically bindsto osteopontin, wherein said antibody comprises a heavy chain amino acidsequence as set forth in SEQ ID NO:11, SEQ ID NO:25, SEQ ID NO:39, SEQID NO:58, SEQ ID NO:62, or SEQ ID NO:66, with the proviso that theC-terminal lysine residue of said heavy chain amino acid sequence isoptionally not present.
 18. The isolated antibody according to claim 17,further comprising a light chain amino acid sequence as set forth in SEQID NO:12, SEQ ID NO:26, SEQ ID NO:40, SEQ ID NO:59, SEQ ID NO:63, SEQ IDNO:67, or SEQ ID NO:78.
 19. An isolated antibody or antigen-bindingportion thereof comprising a V_(H) chain that is encoded by (i) anucleic acid sequence comprising SEQ ID NO:9, SEQ ID NO:23, SEQ IDNO:37, SEQ ID NO:45, SEQ ID NO:49, or SEQ ID NO:53, or (ii) a nucleicacid sequence that hybridizes under high stringency conditions to thecomplementary strand of SEQ ID NO:9, SEQ ID NO:23, SEQ ID NO:37, SEQ IDNO:45, SEQ ID NO:49, or SEQ ID NO:53, wherein said antibody orantigen-binding portion specifically binds to osteopontin.
 20. Theisolated antibody or antigen-binding portion according to claim 19,further comprising a V_(L) chain that is encoded by (i) a nucleic acidsequence comprising SEQ ID NO:10, SEQ ID NO:24, SEQ ID NO:38, SEQ IDNO:47, SEQ ID NO:51, SEQ ID NO:55, or SEQ ID NO:77 or (ii) a nucleicacid sequence that hybridizes under high stringency conditions to thecomplementary strand of SEQ ID NO:10, SEQ ID NO:24, SEQ ID NO:38, SEQ IDNO:47, SEQ ID NO:51, SEQ ID NO:55, or SEQ ID NO:77, wherein saidantibody or antigen-binding portion specifically binds to osteopontin.